The crystal and cryo-EM structures of PLCγ2 reveal dynamic interdomain recognitions in autoinhibition

Young-Cheul Shin; Ashlee Marie Plummer-Medeiros; Alison Mungenast; Hyeong-wook Choi; Karen TenDyke; Xiaojie Zhu; Jennifer Shepard; Kristen Sanders; Ningning Zhuang; Liang Hu; Dongming Qian; Kangkang Song; Chen Xu; John Wang; Suresh B Poda; Maofu Liao; Yu Chen

doi:10.1126/sciadv.adn6037

. 2024 Nov 29;10(48):eadn6037. doi: 10.1126/sciadv.adn6037

The crystal and cryo-EM structures of PLCγ2 reveal dynamic interdomain recognitions in autoinhibition

Young-Cheul Shin ^1,^†,^‡, Ashlee Marie Plummer-Medeiros ^1,^§, Alison Mungenast ^2,^¶, Hyeong-wook Choi ², Karen TenDyke ^2,^#, Xiaojie Zhu ², Jennifer Shepard ^2,^**, Kristen Sanders ², Ningning Zhuang ³, Liang Hu ³, Dongming Qian ³, Kangkang Song ^4,⁵, Chen Xu ^4,⁵, John Wang ², Suresh B Poda ^2,^††, Maofu Liao ^1,^†,^‡,^*, Yu Chen ^2,^*

^¹Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

^²Eisai Inc., 35 Cambridgepark Drive, Cambridge, MA 02140, USA.

^³Viva Biotech Ltd., 735 Ziping Road, Pudong New District, Shanghai 201318, China.

^⁴Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Medical School, Worcester, MA 01605, USA.

^⁵Cryo-EM Core Facility, University of Massachusetts Medical School, Worcester, MA 01655, USA.

Corresponding author. Email: liaomf@sustech.edu.cn (M.L.); julie_chen@eisai.com (Y.C.)

^†

Present address: Department of Chemical Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, Guangdong, China.

^‡

Present address: Institute for Biological Electron Microscopy, Southern University of Science and Technology, Shenzhen, Guangdong, China.

^§

Present address: Bryn Mawr College Chemistry Department, 101 N. Merion Avenue, Bryn Mawr, PA 19010, USA.

^¶

Present address: Delix Therapeutics, 36 Crosby Drive, Bedford, MA 01730, USA.

Present address: 100 Woburn Street, Wilmington, MA 01887, USA.

^**

Present address: 7 Tucker Street, Pepperell, MA 01463, USA.

^††

Present address: Encoded Therapeutics, 341 Oyster Point Blvd., South San Francisco, CA 94080, USA.

Roles

Young-Cheul Shin: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing - original draft, Writing - review & editing

Ashlee Marie Plummer-Medeiros: Methodology, Writing - review & editing

Alison Mungenast: Formal analysis, Investigation, Methodology, Validation, Visualization, Writing - review & editing

Hyeong-wook Choi: Investigation, Writing - review & editing

Karen TenDyke: Formal analysis, Investigation, Validation

Xiaojie Zhu: Investigation

Jennifer Shepard: Conceptualization, Resources

Kristen Sanders: Formal analysis, Investigation, Resources, Validation, Visualization

Ningning Zhuang: Data curation, Formal analysis, Investigation, Validation, Writing - original draft

Liang Hu: Investigation, Resources

Dongming Qian: Investigation, Resources

Kangkang Song: Investigation

Chen Xu: Investigation

John Wang: Supervision, Writing - review & editing

Suresh B Poda: Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Supervision, Validation, Visualization, Writing - review & editing

Maofu Liao: Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing

Yu Chen: Conceptualization, Project administration, Supervision, Visualization, Writing - original draft

PMCID: PMC11606444 PMID: 39612343

Abstract

Phospholipase C gamma 2 (PLCγ2) plays important roles in cell signaling downstream of various membrane receptors. PLCγ2 contains a multidomain inhibitory region critical for its regulation, while it has remained unclear how these domains contribute to PLCγ2 activity modulation. Here we determined three structures of human PLCγ2 in autoinhibited states, which reveal dynamic interactions at the autoinhibition interface, involving the conformational flexibility of the Src homology 3 (SH3) domain in the inhibitory region, and its previously unknown interaction with a carboxyl-terminal helical domain in the core region. We also determined a structure of PLCγ2 bound to the kinase domain of fibroblast growth factor receptor 1 (FGFR1), which demonstrates the recognition of FGFR1 by the nSH2 domain in the inhibitory region of PLCγ2. Our results provide structural insights into PLCγ2 regulation that will facilitate future mechanistic studies to understand the entire activation process.

PLCγ2 structures revealed conformational flexibility of key regulatory domains and found regions involved in autoinhibition.

INTRODUCTION

Phospholipase C gamma 2 (PLCγ2, encoded by gene PLCG2) is a membrane-associated enzyme that catalyzes the conversion of 1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate (PIP₂) to 1D-myo-inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) using calcium as a cofactor. It plays a central role in cellular signaling transduction by generating the key second messenger molecules IP3 and DAG in response to activation by a variety of transmembrane receptors or membrane-associated kinases (1, 2).

Gain-of-function variants in PLCγ2 can cause PLCγ2-associated antibody deficiency and immune dysregulation (PLAID) and autoinflammation and PLCγ2-associated antibody deficiency and immune dysregulation (APLAID) syndromes (1–7). Mouse models of autoimmunity and autoinflammation have also been found carrying gain-of-function missense mutations in PLCγ2 (8, 9). PLCγ2 is a substrate of the Bruton tyrosine kinase (BTK) and mediates its downstream signaling. Activating mutations of PLCγ2 arise in patients with chronic lymphocytic leukemia resistant to the BTK inhibitor ibrutinib (10, 11). Genome-wide association studies also identified a rare variant of PLCγ2 (P522R) associated with reduced risk of Alzheimer’s disease, as well as other dementias including dementia with Lewy bodies and frontotemporal dementia (12–20). Longitudinal analysis also suggested that patients with mild cognitive impairment carrying the P522R variant shows a lower rate of cognitive decline in comparison with noncarriers (21). Activity studies indicated that this variant modestly increases the enzymatic function of PLCγ2 (22–24). Recent studies also suggest that a different missense variant of PLCγ2 (M28L) is associated with loss of function and an elevated risk for Alzheimer’s disease (18, 25, 26).

These observations underscore the importance of finely tuned PLCγ2 regulation. PLCγ enzymes are unique in the PLC family for containing multiple folded domains in the inhibitory region. The previously reported crystal structure of rat PLCγ1 (rPLCγ1) provides insights into how the inhibitory region associates with the core region and occludes the access of the catalytic site to substrate (27). It will be helpful to observe similar atomic details for human PLCγ2 and further address whether additional interactions may be involved in this autoinhibitory interaction.

Similar to PLCγ1 (28, 29), PLCγ2 is regulated by receptor tyrosine kinases and transmembrane receptor–associated kinases by phosphorylation at key regulating tyrosine residues (1, 2). One of the most comprehensive studies of this regulation for PLCγ enzymes pertains to growth factor receptor tyrosine kinases phosphorylation (30–32). Fibroblast growth factor receptor 1 (FGFR1) phosphorylates PLCγ enzymes, which contribute to the alleviation of PLCγ autoinhibition and recruitment of PLCγ to the vicinity of the membrane where its substrate PIP₂ resides (33, 34). However, limited knowledge is available to help understand the recognition of the intact PLCγ enzyme by regulatory kinases and whether these associations alone are sufficient to trigger the major conformational changes leading to PLCγ activation.

In this study, we determined crystal and cryo–electron microscopy (cryo-EM) structures of human PLCγ2 in autoinhibited conformations (referred to as PLCγ2-C, PLCγ2-F, and PLCγ2-H), which revealed multiple recognition sites between domains in the inhibitory region and the core region. We also conducted cryo-EM analysis of PLCγ2 in complex with phosphorylated FGFR1 kinase, which confirmed that the FGFR1 kinase domain recognizes the nSH2 domain of PLCγ2 in an autoinhibition state. Our data provide a view of dynamic PLCγ2 autoinhibition, which is likely important for PLCγ2 fine regulation and applicable to PLCγ1 as well.

RESULTS

Crystal structure of human PLCγ2

Multiple constructs containing N-terminal, C-terminal, or internal deletions were explored to enable protein crystallization. One construct, PLCγ2-C (14-221-GSG-239-1190; Fig. 1A), resulted in diffracting crystals. This construct contains all known structural domains of PLCγ2 (Fig. 1A, amino acid 14-1190), and a glycine-serine-glycine (GSG) peptide replacing a loop (amino acid 222-238) in the predicted EF-Hand domain.

A crystal structure of human PLCγ2 using this construct was determined at 2.55-Å resolution (Fig. 1B and table S1). This particular crystal was soaked with a compound overnight before diffraction data collection. However, since no extra densities of compound were observed and the structure is identical to another one obtained without compound soaking at 2.75-Å resolution, we determined that this structure at 2.55-Å resolution represents an apo structure. As it exhibited improved resolution over the structure determined at 2.75-Å resolution, we report and focus on description of this structure in this section.

The atomic model contains all domains except for the nSH2 domain, for which very limited density was observed. In this structure, the membrane engagement side of the core region is blocked by the regulatory region, indicating that it represents the autoinhibited state. The sequence connecting the cSH2 and Src homology 3 (SH3) domain (amino acids 754 to 766, Y759 linker) is clearly visible in this structure (fig. S1A), running toward the interface between the inhibitory and core region and interacting with the spPH domain (fig. S1B). This sequence contains a key regulatory tyrosine Tyr⁷⁵⁹, and the phosphorylation of Tyr⁷⁵⁹ has been shown to be closely related with the activation of PLCγ2 (35–37). To mimic the potential impact of Tyr⁷⁵⁹ phosphorylation alone, we generated a phosphor-mimetic version of PLCγ2 by replacing Tyr⁷⁵⁹ with a glutamate residue. Recombinant protein of PLCγ2 Y759E exhibited enhanced activity in comparison with the wild-type PLCγ2 in a biochemical lipase assay (fig. S1C) (38). These results support that phosphorylation of Tyr⁷⁵⁹ alone would be important for PLCγ2 activation.

Similar to the previously reported autoinhibited structure of rPLCγ1 [Protein Data Bank (PDB): 6PBC (27)], spPH and cSH2 domains in this structure of PLCγ2 directly interact with the TIM barrel (TIM-X/Y) and C2 domain, respectively, forming major interfaces between the inhibitory and the core regions (Fig. 1, C and D). In addition, the SH3 domain is in proximity to and interacts with both the TIM barrel and C2 domain in the core region via the linker between the SH3 and spPH domains (SH3-spPH linker) and the SH3 N-terminal loop that follows the Y759 linker (SH3 N-loop), respectively (Fig. 1E). These interactions contribute to the interfaces between the inhibitory and the core regions and may play a role in the autoinhibition regulation as well. A direct association of the SH3 domain with the core region was not observed in the previously reported rPLCγ1 structure, in which the SH3 domain was orientated away from the inhibitory-core region interface and located on the outmost side of the inhibitory region (fig. S2). This may be due to the replacement of the 25-residue loop connecting cSH2 and SH3 domains by a short linker of Ser-Gly-Ser in the rPLCγ1 structure described (27). The interface analysis between the inhibitory and core regions of human PLCγ2 suggests that three domains from the inhibitory region, spPH, cSH2, and SH3, are directly involved in the interface interaction and probably engaged with PLCγ2 autoinhibition regulation.

Disease-associated mutations and variants of PLCγ2

Disease-associated mutations and variants of PLCγ2 have been noted in both inhibitory and core regions, while most have been reported in the inhibitory region and most of them are gain-of-function mutations (Fig. 2A). When mapping the sites of gain-of-function point mutations in the autoinhibited conformation shown by the crystal structure of PLCγ2 (Fig. 2B), most are localized at the interface between the inhibitory and core regions, including both the cSH2/C2 and SH3-spPH linker/TIM barrel interaction sites. This observation suggests that both interaction sites are critical for PLCγ2 autoinhibitory regulation and that mutations at either site may destabilize the inhibitory-core region interface and result in increased basal enzymatic activity. Similarly, the gain-of-function mutations harbored by two autoinflammatory disease mouse models, D993G in Ali5 mice and Y495C in Ali14 mice, are also located at the interface between the inhibitory and core regions, specifically the SH3-spPH linker/TIM interaction site (figs. S3 and S4A). We conducted IP One assays to examine the enzymatic activity of selected mutations and confirmed that they enhanced basal PLCγ2 phospholipase activity (fig. S4B).

Fig. 2. — (A) Positions of gain-of-function point mutations are indicated by red arrows, and the deletions are denoted by red diagonal stripes. Loss-of-function mutation is noted by a dark blue arrow. Disease association is illustrated by filled circles: yellow, APLAID; turquoise blue, PLAID; green, Ibrutinib resistance; purple, Alzheimer’s disease. (B) Mapping of PLCγ2 gain-of-function mutations onto the crystal structure of PLCγ2. Residues at the mutation site are colored in red and shown in sphere representation to label the position of mutation. Label denotes mutations found so far at each position. Disease association is illustrated by filled circles as in (A). Sites for P552R and S1192G are not shown as residues Pro⁵²² and Ser¹¹⁹² are invisible or absent in the crystal structure, respectively.

When mapping the reported deletions in the crystal structure of PLCγ2, the deletion linked to APLAID (Δ845-848) is located within the SH3-spPH linker in direct association with the TIM-barrel catalytic domain in the core region (fig. S4C and Fig. 1E). This deletion has been shown to greatly enhance PLCγ2 activity (5), likely due to the disruption of autoinhibitory recognition at this site. As expected, the causative deletions for PLAID (exon 19 and exon 20-22 deletions: Δ19 and Δ20-22) result in the loss of the most of cSH2 domain (amino acids 646 to 685) and most of cSH2-Y759 linker-SH3 domains (amino acids 686 to 806), respectively, which are expected to have a major impact on the integrity of autoinhibitory interface and result in higher basal phospholipase activity (fig. S4C).

Our analysis suggests that the inhibitory-core region interface has crucial functions in PLCγ2 autoinhibition regulation and activity modulation. The recognition sites between the cSH2 and C2 domains, and between SH3-spPH and TIM barrel, respectively, play indispensable roles.

Structure analysis by single-particle cryo-EM

To capture additional structure details in full-length PLCγ2 and gain insights into PLCγ2 recognition by FGFR1, we generated recombinant proteins of human PLCγ2 with the complete sequence (Fig. 1A, amino acids 1 to 1265) and the kinase domain (amino acids 456 to 774) of human FGFR1 (FGFR1K). The association between PLCγ2 and FGFR1K was examined by size exclusion chromatography in the presence and absence of FGFR1K phosphorylation of the key regulating tyrosine Tyr⁷⁶⁶. Both full-length PLCγ2 and PLCγ2-C were tested. The results demonstrated that phosphorylated FGFR1K (pFGFR1K) interacts and co-elutes with PLCγ2 in both cases, while FGFR1K lacking phosphorylation is largely eluted separately from PLCγ2, confirming that phosphorylation of FGFR1K is required for associating with PLCγ2 (fig. S5) as previously reported (33, 34).

For cryo-EM analysis, the full-length PLCγ2 was incubated with pFGFR1K. A three-dimensional (3D) reconstruction of PLCγ2 was obtained at 3.7-Å resolution (referred to as structure PLCγ2-F; fig. S6), but the SH3 and C-terminal regions of PLCγ2 and FGFR1K were not well resolved. To improve the density, the particle set was further processed in two directions (fig. S6). First, we performed masked 3D classification without alignment in Relion and local refinement in cryoSPARC, obtaining a 4.1-Å resolution map of PLCγ2 with clear density of the C-terminal region (referred to as structure PLCγ2-H). Second, we generated a complex mask covering both the densities of PLCγ2 and FGFR1K, obtaining a full complex map at 4.0-Å resolution (referred to as structure PLCγ2/pFGFR1K) by performing 3D classification without alignment and local refinement (in Relion and in cryoSPARC, respectively). Three cryo-EM structures involving human PLCγ2 were determined (fig. S6 and table S2).

Cryo-EM structures of PLCγ2

All major domains of PLCγ2 were mostly well resolved in the cryo-EM density map of PLCγ2-F, including the nSH2 domain in the inhibitory region that was missing in the crystal structure (Fig. 3A and fig. S6). The nSH2 domain is connected to the cSH2 domain and positioned to readily bind to upstream kinases and recruit interacting partners, as previously observed in the crystal structure of rPLCγ1 (27). Overlay of the cryo-EM structure of PLCγ2 with the crystal structure of rPLCγ1 revealed a ~20° shift of the nSH2 domain (fig. S7A), which may be related to its conformational flexibility relative to the rest of the protein.

Fig. 3. — (A) Cryo-EM map of PLCγ2-F (3.7-Å resolution) presented in both front (left) and back (right) views. Domains are colored as in Fig. 1A, and the nSH2 domain (in light blue) is visible. (B) Structural model of PLCγ2 with complete inhibitory region. Left, crystal structure of PLCγ2 contains the entire SH3 domain and Y759 linker (highlighted in orange circle) but not the nSH2 domain. Middle, cryo-EM structure of full-length PLCγ2 (PLCγ2-F) has visible nSH2 domain (highlighted in green circle) but misses the vast majority of SH3 and Y759 linker. Right, a structural model of PLCγ2 containing all inhibitory domains was built by overlaying domains visible in both structures. Core region is shown in surface representation in gray. Inhibitory region domains are shown in carton representation and colored as in Fig. 1A.

The cryo-EM map of PLCγ2-F contains weak densities for the SH3 domain and the Y759 linker connecting cSH2 and SH3 domains, indicating that this region may be conformationally flexible. Domains visible in both crystal and this cryo-EM structure of PLCγ2 are well overlayed (root mean square deviation = 1.262 Å), which allows us to build a structure model containing all inhibitory domains, providing a comprehensive view of PLCγ2 in the autoinhibited state (Fig. 3B). In this model, the membrane-associating side of the core region is engaged in direct interactions with three of the four major domains in the inhibitory region (spPH, SH3, and cSH2), while the fourth domain, the nSH2 domain, is free from the interface engagement and accessible for recognition with interacting partners.

The cryo-EM map of PLCγ2-H contains strong densities for the SH3 domain as well as extra helical densities that were not explained by the known domains from PLCγ2 (Fig. 4A and fig. S6). The extra densities connect to and follow the C-terminal end of the C2 domain in the core region, indicating that they correspond to the very C-terminal sequence of the full-length protein following the C2 domain (Fig. 1A). The helical conformation of this region was also predicted by AlphaFold (fig. S7B) (39, 40), in agreement with our analysis. An atomic model was built into the visible densities in this region, suggesting that the C-terminal sequence 1189 to 1259 forms a helix-turn-helix structure (Fig. 4A). As this region has not been structurally annotated to our knowledge, we named this structure the H domain (Figs. 1A and 4A). In this cryo-EM structure, the H domain folds back to the rest of the protein and contacts the SH3 domain, suggesting a role for such an interaction in regulating PLCγ2 autoinhibition and activation.

Superimposition of the cryo-EM structure PLCγ2-H with the crystal structure PLCγ2-C revealed conformational changes in the SH3 domain (Fig. 4B). Although the structural fold of the N-terminal region of the SH3 domain (amino acids 767 to 828) remains unchanged, it shifts and rotates toward the H domain in PLCγ2-H. The C-terminal helical region of the SH3 domain (amino acids 829 to 846) also rotates and displays a more extended conformation in PLCγ2-H. The N- and C- terminal regions of the SH3 domain are ~90° relative to each other in PLCγ2-C, while this angle is extended to ~120° in PLCγ2-H (Fig. 4B). This observation is consistent with the conformational flexibility of SH3 domain as noted in the structure PLCγ2-F, which may contribute to the direct association between the SH3 and H domains and be relevant to its role in PLCγ2 activity modulation. The crystal and cryo-EM structures described so far likely represent different autoinhibited conformations of human PLCγ2, and we decided to further examine this in the presence of interacting partners.

Cryo-EM structure of PLCγ2 in complex with FGFR1K

The cryo-EM density map of PLCγ2/pFGFR1K was refined to 4.0-Å average resolution and confidently demonstrated the presence of FGFR1K in the complex (Fig. 5A and fig. S6). The recognition of FGFR1K by PLCγ2 is mainly mediated by the nSH2 domain in a similar orientation as previously reported crystal structure of FGFR1K bound to rPLCγ1 nSH2 [PDB: 3GQI (31)]. Conformational flexibility of nSH2 domain was observed when comparing PLCγ2 structures obtained so far, which may facilitate its recognition by regulating partners (fig. S7A).

Fig. 5. — (A) Cryo-EM map of human PLCγ2/pFGFR1K complex (4.0-Å resolution) presented in both front (left) and back (right) views. PLCγ2 domains are colored as in Fig. 1A, and FGFR1K is colored in orange. (B) Structures of PLCγ2 and PLCγ2/pFGFR1K complex. All shown in cartoon representation and colored as in Fig. 5A. Active site of FGFR1K is noted by a black circle in dashed line. The distance between Y753 in the cSH2-SH3 linker and the active site of FGFR1K is noted by a dashed arrow. PLCγ2 cryo-EM structure only shows PLCγ2-F for illustration.

PLCγ2 exhibited an autoinhibited conformation in this complex structure, similar to the conformation observed in the PLCγ2 alone crystal and cryo-EM structures (Fig. 5B). This suggests that the physical association of FGFR1K with PLCγ2 alone may not induce a prominent conformational change in PLCγ2. Previous cryo-EM analysis of a cross-linked PLCγ1-FGFR1K complex also showed an autoinhibited state of PLCγ1 in the complex (30). Key regulatory residues Tyr⁷⁵³ and Tyr⁷⁵⁹ of PLCγ2 are expected to be phosphorylated by regulating kinases, which may contribute to subsequent autoinhibition relief and enzyme activation. Tyr⁷⁵⁹ was not visible in the cryo-EM structure of PLCγ2/pFGFR1K, making it difficult to evaluate how this residue may be accessible to the active site of FGFR1K. Tyr⁷⁵³ is visible in this structure and is about 70 Å in linear distance to the active site of FGFR1K (Fig. 5B). On the basis of this observation, Tyr⁷⁵⁹ and Tyr⁷⁵³ are unlikely directly accessible to the active site of FGFR1K, and additional conformational changes would be needed to allow the phosphorylation of either tyrosine by bound FGFR1. As endogenous FGFR1 is a transmembrane protein located on the plasma membrane and PLCγ2 typically executes enzymatic activity on the membrane, it may be critical to fully capture the modulation of PLCγ2 by FGFR in the presence of relevant membrane environments. Our results suggest that pFGFR1K recognizes the nSH2 domain in the autoinhibited PLCγ2, which may require additional conformational modulation to enable the phosphorylation of key regulating tyrosine residues and thus activate PLCγ2.

DISCUSSION

In comparison with other members of the phospholipase C family, the PLCγ enzymes contain a structurally complicated inhibitory region. Our results suggest that most of these domains, except for the nSH2 domain, are directly involved in the association with the core region in the autoinhibited states. The structures we determined exhibit a wide range of conformations that probably exist during autoinhibition, indicative of dynamic interdomain interactions at the interface between the inhibitory and core regions that likely contribute to the regulation of PLCγ2 by different processes (Fig. 6, left). Two recognition areas, the site involving SH3-spPH from the inhibitory region and the TIM barrel catalytic core, and the interface between the cSH2 domain in the inhibitory region and C2 domain in the core region, are present in all autoinhibited structures, suggesting that they may be required for the enzyme in the fully closed autoinhibition state. Most of the gain-of-function mutations are also located in these areas, so supporting the integrity of both recognition sites may be critical for complete inhibition of PLCγ enzymes (Fig. 2B) (27).

Fig. 6. — The autoinhibited states of PLCγ2 (left) exist in multiple conformations, exhibited by structural flexibility of the SH3 and C-terminal H domains. The recognition sites between cSH2 and C2 domains, and between SH3-spPH linker and TIM barrel, respectively, play indispensable roles. Recognition of PLCγ2 by FGFR1K (middle) is mediated by the nSH2 domain in the inhibitory region, and FGFR1K phosphorylation dependent. This recognition may position PLCγ2 in proximity to the membrane as well as a second FGFR1K nearby, both of which may be involved in the next step of phosphorylation and conformation change of PLCγ2, resulting in the disengagement of the inhibitory region and access of catalytic core to PIP₂ substrate membrane (right). Core region is shown in surface representation in gray, in which the TIM barrel that harbors the catalytic center is colored in dark gray. Newly identified H domain is shown in cartoon representation in red. Inhibitory region domains are illustrated in color blocks and colored as in Fig. 1A. FGFR1K associated with PLCγ2 is presented in surface representation in orange, and the rest of the molecule and a second copy of FGFR1 are shown in orange shape blocks. The phosphorylated site of FGFR1K is denoted by “P” in red circle. Plasma membrane bilayer is shown in light gray, and a substrate PIP₂ is highlighted in green.

Both the SH3 domain in the inhibitory region and the newly observed H domain in the C-terminal region of the core domain contribute to the observed structural dynamics in PLCγ2 (Fig. 6, left). The SH3 domain exhibited different conformations among the three PLCγ2 structures we present here. Limited densities are observed for this domain in PLCγ2-F, while clear densities are visible in both PLCγ2-C and PLCγ2-H structures, which may be facilitated by the crystal contact and the interaction with the H domain, respectively (fig. S8, A to C). These observations suggest that the SH3 domain is conformationally flexible. It interacts with different domains from the core region and is covalently connected to the regulatory Y759 linker and catalytic core-covering spPH domain, indicating that the SH3 domain may play an important role in connecting different domains during the PLCγ2 autoinhibition regulation. This may be applicable to PLCγ1 as well. It remains to be investigated mechanistically how the conformational flexibility of the SH3 domain directly affects the activity regulation of PLCγ enzymes. One structure from our cryo-EM studies, PLCγ2-H, also showed structural details of the C-terminal region of PLCγ2. It exhibited a helix-turn-helix structure and folds back to the rest of the core region, engaging directly in interactions with the SH3 domain from the inhibitory region. We named this segment the H domain to reflect its structural features, and the structure indicates that this newly described domain may also participate in autoinhibition and perhaps activity regulation of PLCγ2. One of the reported ibrutinib-resistant mutation S1192G (11), is localized in this segment. Deletion of the H domain or S1192G mutation exhibited limited impact on PLCγ2 activity biochemically (fig. S8D). Studies are still needed to elucidate how PLCγ2 H domain and its recognition to the inhibitory region contribute to the enzymatic activity regulation in cells. It remains unclear whether similar structures exist at the C-terminal region of PLCγ1. This segment has moderate sequence similarity between human PLCγ1 and PLCγ2 (~17% sequence identity; fig. S3), and in silico predictions suggests a high likelihood of disorder in this segment of PLCγ1.

PLCγ enzymes can be activated downstream of activated FGFR signaling, while the mechanistic understanding of this process is still missing. The cryo-EM structure of PLCγ2 in complex with the phosphorylated kinase domain of FGFR1 shows that their recognition is mediated by the nSH2 domain in the inhibitory region, consistent with similar reports for PLCγ1 (30, 31). This association has a very limited impact on the overall conformation of PLCγ2, suggesting that this may represent the first step of recognition (Fig. 6, middle). This will also likely bring PLCγ2 in close proximity to the inner leaflet of the plasma membrane, where its substrate PIP₂ resides.

It also remains unclear how FGFR1K phosphorylates the key regulating tyrosine residues in PLCγ enzymes. Our cryo-EM structure of PLCγ2/pFGFR1K suggests that these residues remain far from the active site of the associating FGFR1K (Fig. 5B). As FGFR1R dimerizes upon activation, there may be a possibility for the involvement of the kinase domain from the other copy of FGFR1 in getting access to and modifying these key tyrosine residues located in the Y759 linker between the cSH2 and SH3 domains in the inhibitory region (Fig. 6, middle). Upon phosphorylation, this linker may disengage from its interaction with the spPH domain and induce further conformational changes to remove the inhibitory region from the membrane-associating side of the core region (Fig. 6, right). In this case, the proximity of PLCγ2 to the plasma membrane as a result of the FGFR1K-PLCγ2 interaction may additionally facilitate the direct association of the membrane with the expected lipid-associating domains in the core region (PH, EF-Hand, and C2 domains), which will further enhance the anchoring of PLCγ2 to the membrane and its access to the PIP₂ substrate. Our studies provide structural insights into PLCγ2 autoinhibition and upstream factor recognition, which may have general mechanistic implications for PLCγ enzymes in healthy and disease states.

The structures described in this study were all obtained in membrane-free conditions, which may have limitations in presenting conformation(s) in proximity to and/or in association with the membrane. Future structure studies of human PLCγ2 and its association with interacting partners in the presence of membrane will provide additional insights. The study presented also revealed conformational flexibility of the regulator domain SH3 and found a C-terminal structure (H domain) involved in the autoinhibition interface, while it remains unclear how they directly affect the regulation of PLCγ2 activity. Future mechanistic studies through mutagenesis, cross-linking, and molecular dynamics will contribute to the understanding.

MATERIALS AND METHODS

Experimental design

This study aims to gain structure insights of human PLCγ2, alone and when in complex with an interacting partner, by combinatory approaches of x-ray crystallography and single-particle cryo-EM.

Protein cloning, expression, and purification

The human PLCγ2 construct PLCγ2-C (14-221-GSG-239-1190) was generated to facilitate protein crystallization, and cloned into a modified pFastBacHT vector, pFBLIC, which incorporates a His6 tag at the N terminus of the expressed protein cleavable by Tobacco Etch Virus (TEV) protease, using a previously published ligation-independent cloning strategy (41). The construct was transformed into DH10Bac cells (Invitrogen) to produce bacmid DNA, which was subsequently used to generate baculovirus in Sf9 cells according to the manufacturer’s protocol (Invitrogen Bac-to-Bac manual). High Five insect cells at a density of 2.0 × 10⁶ cells/ml were infected with baculovirus encoding PLCγ2-C at a multiplicity of infection of ~5.0 for 48 hours at 27°C. Cells were harvested at 2000 rpm and resuspended in lysis buffer containing 25 mM tris (pH 7.5), 300 mM NaCl, 5% glycerol, and 1 mM TCEP and supplemented with EDTA-free complete protease inhibitor tablets (Roche Applied Science). Cells were sonicated and then spun at 12000 rpm for 1 hour at 4°C. The clarified supernatant was loaded onto a Ni–nitrilotriacetic acid (NTA) column (Qiagen) that equilibrated with lysis buffer. The column was washed with 20 column volumes of the buffer containing 20 mM imidazole and then eluted with 20 column volumes of the buffer containing 250 mM imidazole. Eluted PLCγ2 protein was dialyzed overnight against lysis buffer in the presence of the TEV protease to remove the His6 tag. Cleaved protein was then subjected to a second passage over a Ni-NTA column. Fractions containing purified proteins were concentrated and further purified by a Superdex 200 size-exclusion column (GE Healthcare) equilibrated in buffer containing in the 25 mM tris (pH 7.5), 150 mM NaCl, 1 mM TCEP, and 5% glycerol.

For cryo-EM and biochemical studies, the DNA sequence encoding full-length human PLCγ2 (1-1265) was cloned into the pTT5 expression vector with N-terminal Flag tag to facilitate protein purification. The construct was transiently transfected into Expi293F cells (Thermo Fisher Scientific). The cells were cultured at 37°C in reduced-serum medium (Gibco) with 8% CO₂ at a speed of 125 rpm, and cells were collected 72 hours after transfection. The cells were harvested at 2000 rpm and resuspended in lysis buffer containing 25 mM tris (pH 7.5), 300 mM NaCl, 5% glycerol, and supplemented with EDTA-free complete protease inhibitor tablets (Roche Applied Science). Cells were sonicated and then spun at 12000 rpm for 1 hour at 4°C. The clarified supernatant was captured by anti-Flag M2 affinity gel resin (Sigma-Aldrich), followed by size-exclusion chromatography purification using a Superdex 200 column (GE Healthcare) equilibrated in buffer containing 25 mM tris (pH 7.5), 150 mM NaCl, 1 mM TCEP, and 5% glycerol. The recombinant protein of human PLCγ2 Y759E, delH (1-1189), and S1192G were generated using the same procedure.

To generate FGFR1 kinase domain recombinantly, plasmid DNA encoding the kinase domain (residues 456 to 774) of human FGFR1 with six substitutions (Y463F, Y583F, Y585F, Y653F, Y654F, and Y730F) (42) was cloned into pET28a vector incorporating a His6-tag and a SUMO tag at the N terminus of the expressed protein and expressed in Escherichia coli BL21 gold strain (Novagen). Cells were grown at 37°C in TB medium containing kanamycin (0.1 mg/ml) and tetracyline (0.050 mg/ml) to an optical density at 600 of ~3.0. Protein expression was induced for 20 hours at 16°C with 0.1 mM isopropyl β-d-thiogalactoside (final concentration). The cells were collected by centrifugation and resuspended in lysis buffer containing 25 mM tris (pH 7.5), 300 mM NaCl, 5% glycerol, and 1 mM TCEP. The cells were lysed using high-pressure homogenizer, and CHAPS was added to a final concentration of 0.5% w/v followed by incubation at 4°C for 30 min. Soluble lysate was prepared by centrifugation, and the kinase domain of FGFR1 was isolated by Ni-NTA column (Qiagen). The eluted protein was dialyzed overnight against lysis buffer in the presence of the ULP1 protease to remove the His-SUMO tag and then subjected to a second passage over a Ni-NTA column. The unbound protein was further purified by ion-exchange chromatography using a HiTrap Q-HP 5-ml column (Cytiva), and the phosphorylated kinase domain of FGFR1 was collected after size exclusion chromatography purification using Superdex 75 10/300 GL column (GE Healthcare). To prepare the dephosphorylated form, purified phosphorylated kinase domain of FGFR1 was treated with His-tagged lambda phosphatase in the presence of 2 mM MnCl₂ at 4°C overnight, followed by isolation using Ni-NTA column and purification by size exclusion chromatography (Superdex 75 column). Intact protein molecular weight was detected by liquid chromatography–mass spectrometry using an Agilent 6530 quadrupole orthogonal acceleration–time-of-flight mass spectrometry connected to an Agilent 1290 UPLC, confirming the phosphorylation status. The phosphorylation site in the phosphorylated kinase domain of FGFR1K was detected at Tyr⁷⁶⁶ by protein mapping.

Protein concentrations were determined using the A280 method with extinction coefficients calculated using the ProtParam tool (ExPaSy Molecular Biology Server (43)). The prepared protein was further analyzed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) to confirm concentration and purity and subsequently stored at −80°C after flash freezing in liquid nitrogen.

Crystallization

Crystals of PLCγ2 were grown initially by sitting drop vapor diffusion using purified protein from construct PLCγ2-C. Protein solution was prepared at 10 mg/ml in buffer containing 20 mM Hepes (pH 7.5), 150 mM NaCl, 1 mM TCEP, and 5% glycerol. Two hundred nanoliters of this protein solution was mixed with 200 nl of reservoir solution that contains 0.1 M magnesium formate and 15% w/v polyethylene glycol (PEG) 3350 and equilibrated against a 40-μl reservoir. Crystals appeared after one week at 18°C. Diffraction quality crystals were grown at 18°C by microseeding hanging drops. Drops were prepared by mixing 1 μl of reservoir solution (0.1 M magnesium formate, 15% w/v PEG 3350) with 1 μl of protein solution. Drops were equilibrated against a 500-ml reservoir. Crystals were grown to ~100 μm on the longest edge after 4 to 5 days. Some of them were also transferred to a new 2-μl hanging drop with a reservoir solution (0.1 M magnesium formate, 15% w/v PEG 3350) for overnight compound soaking. The crystals were cryoprotected in mother liquor containing 20% ethylene glycol and flash-frozen in liquid nitrogen on nylon loops.

Diffraction data collection and crystal structure determination

The diffraction data were collected at Shanghai Synchrotron Radiation Facility [BL19U1 (44)] and processed using autoPROC (45) that includes XDS (46) and AIMLESS (CCP4 package) (47). The initial molecular replacement was performed by Phaser MR (48) with rPLCγ1 core domain (PDB code: 6PBC) as the template. Structure refinement was carried out using Refmac5 (49) combined with several rounds of COOT (50) manual fitting. The calcium ion and antimony ion were accurately fit into the positive density maps according to the electron density maps after an initial round of refinement. Figures were prepared with PyMOL (The PyMOL Molecular Graphics System) (51). Complete data collection and refinement statistics are shown in table S1.

Cryo-EM sample preparation and data collection

The final preparation of the PLCγ2 sample was carried out using a Superose 6 Increase 10/300 column, which was equilibrated with 25 mM tris (pH 7.9) and 150 mM NaCl. Fractions were collected, concentrated to 1 mg/ml, and then mixed with FGFR1K (5 mg/ml) at a 1:18 molar ratio. Following a 30-min incubation, the sample was further concentrated to 1.06 and 2.7 mg/ml for cryo-EM grid preparation. A volume of 3.5 μl of the prepared sample was applied to a glow-discharged holey grid (Quantifoil Au300 R1.2/1.3) and then blotted for 3.5 s at room temperature with 90% humidity using a Gatan Cryoplunge 3 system. The samples were then vitrified in liquid ethane cooled by liquid nitrogen. Cryo-EM images were collected on a Talos Arctica (Thermo Fisher Scientific). Additional details are provided in table S2.

Cryo-EM data processing

Detailed information regarding the number of particles and map resolutions can be found in table S2 and fig. S6. Image sets from two different samples were combined, consisting of 3369 images from the 1.06 mg/ml sample and 2974 images from the 2.7 mg/ml sample. Movies were motion corrected with MotionCor2 (52) and defocus values calculated with CTFFIND4 (53). All initial 2D processing steps were performed using the SAMUEL (Simplified Application Managing Utilities for EM Labs) Package (54). Dose-weighted images were binned three times with “sampilcopy2d.py” and interactively screened using “SamViewer.” Particle picking, particle stack generation, star file generation, and 2D classification were performed using “samautopick.py,” “sampilboxparticle.py,” “samrelion.py new,” and “samtree2dv3.py,” respectively. Additional 2D classification from individual 2D classes was performed using “samrelion.py 2dcls,” which used RELION 3.0, and 3D classification was carried out in RELION 3.0 (55).

For mask generation, the best maps from 3D classification were low-pass filtered to 30-Å resolution using “relion_image_handler” and masks were generated using “relion_mask create” in RELION with 2-pixel extension and 6 pixels for smoothening of the edges. Further refinement for obtaining final maps was carried out using “Local refinement” in cryoSPARC (56). The resolutions were estimated on the basis of the FSC=0.143 criterion, and the curves were calculated using “Comprehensive Validation” in Phenix (57). Last, the processed maps were obtained for analysis, and the resulting structures were referred to as PLCγ2-F, PLCγ2-H, and PLCγ2-FGFR1K, as described in fig. S6.

Cryo-EM structure model building, refinement, and display

Three structure models (PLCγ2-F, PLCγ2-H, and PLCγ2/pFGFR1K) from cryo-EM data were built using Alphafold2 models of PLCγ2 and FGFR1K as the starting templates. SH2, SH3, and C-terminal helix domains were extracted using PyMOL (Version 2.5.2, Schrödinger LLC.), and all fragmented domains were fitted to the maps using the “fit in map” function in UCSF ChimeraX (58). Any disconnected loops, side-chain rotamers, varied secondary structures, or unmatched regions to the map were manually corrected at sigma levels of 3.0~7.0 using “Real space refine zone” in Coot (59). The models were further refined using “real-space refinement” in Phenix. Final models were validated using “Comprehensive validation” in Phenix after outlier correction in Coot. Figures of structural models and cryo-EM maps were generated using UCSF ChimeraX and PyMOL.

Size exclusion chromatography examining PLCγ2/FGFR1K association

Phosphorylated and dephosphorylated FGFR1K were mixed in equal molar ratio with PLCγ2 (full-length PLCγ2 and PLCγ2-C) respectively in the presence of 5 mM MgCl₂ and incubated at 4°C overnight. The mixtures were then loaded onto a Superdex 200 10/300 GL size exclusion column equilibrated with buffer containing 25 mM tris (pH 7.5), 150 mM NaCl, 5 mM MgCl₂, 5% glycerol, and 1 mM TCEP, and the elution fractions were analyzed by SDS-PAGE.

Lipase activity measurement

Lipid vesicles containing the fluorogenic substrate XY-69 was prepared as previously described (38, 60). Briefly, lipid vesicles were generated containing 0.46 μM XY-69, 57.6 μM PIP2, and 230 μM phosphatidylethanolamine. In each reaction, 10 μl of lipid vesicle solution was mixed with 2 μl of recombinant PLCγ2 (3.13 ng in wild-type and Y759E PLCγ2 comparison and 1.6 ng in wild-type, delH, and S1192 PLCγ2 examination), and the fluorescence was read immediately and recorded at 1- to 2-min intervals for 64 min (485-nm excitation and 520-nm emission).

The assessment of PLC activity using the IP One assay was carried out by cotransfecting human embryonic kidney 293 cells (PLCG1 knock-out) with constructs expressing PLCG2 variants and the cofactor Rac2^G12V (61). The assay was conducted using the IP-One Gq kit (PerkinElmer) according to the protocol. Homogeneous Time Resolved Fluorescence (HTRF) signal was read by Spark 20M microplate reader (Tecan, 337-nm excitation and 620- and 665-nm emission).

Acknowledgments

We gratefully acknowledge J. Sondek, N. Hajicek, and S. Endo-Streeter for the helpful discussion of PLCγ structure features and FGFR regulation, and advice of FGFR construct design. We gratefully acknowledge B. Cravatt and H. Jing for the helpful discussion and suggestions for the IP One assay. We gratefully acknowledge Q. Zhang for the helpful suggestion of phospholipase activity assay using XY-69.

Funding: This research was funded by Eisai Inc.

Author contributions: Conceptualization: Y.C. and M.L. Investigation: Y.-C.S., A.M.P.-M., A.M., H.C., K.T., X.Z., J.S., K.Sa., N.Z., L.H., D.Q., K.So., and C.X. Analysis: Y.-C.S., M.L., A.M., K.T., K.Sa., and Y.C. Data curation Y.-C.S., M.L., and Y.C. Visualization: Y.-C.S., A.M., K.T., K.Sa., L.H., and Y.C. Supervision: Y.C., M.L., and J.W. Writing—original draft: Y.-C.S., M.L., and Y.C. Writing—review and editing: Y.-C.S., A.M.P.-M., A.M., H.C., K.T., X.Z., J.S., K.Sa., N.Z., L.H., D.Q., K.So., C.X., J.W., S.B.P., M.L., and Y.C.

Competing interests: A.M., H.C., K.T., X.Z., J.S., K.Sa., J.W., S.B.P., and Y.C. are/were employees of Eisai Inc. The other authors declare that they have no competing interests.

Data and materials availability: The atomic coordinates and structure factors for the crystal structure of human PLCγ2 (PLCγ2-C) have been deposited in the Protein Data Bank (code 8T7C, http://wwpdb.org/). Structure coordinates for the cryo-EM structures of full-length PLCγ2 (PLCγ2-F), PLCγ2 containing visible H domain (PLCγ2-H), and PLCγ2 in complex with pFGFR1K (PLCγ2/pFGFR1K) have been deposited in the Protein Data Bank under entry codes 8JQG, 8JQH, and 8JQI, respectively. The corresponding cryo-EM maps are deposited in the Electron Microscopy Data Bank under the accession numbers EMD-36571, EMD-36572, and EMD-36573. All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.

Supplementary Materials

This PDF file includes:

Figs. S1 to S8

Tables S1 and S2

sciadv.adn6037_sm.pdf^{(4.1MB, pdf)}

REFERENCES AND NOTES

1.Jackson J. T., Mulazzani E., Nutt S. L., Masters S. L., The role of PLCγ2 in immunological disorders, cancer, and neurodegeneration. J. Biol. Chem. 297, 100905 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Koss H., Bunney T. D., Behjati S., Katan M., Dysfunction of phospholipase Cγ in immune disorders and cancer. Trends Biochem. Sci. 39, 603–611 (2014). [DOI] [PubMed] [Google Scholar]
3.Zhou Q., Lee G. S., Brady J., Datta S., Katan M., Sheikh A., Martins M. S., Bunney T. D., Santich B. H., Moir S., Kuhns D. B., Priel D. A. L., Ombrello A., Stone D., Ombrello M. J., Khan J., Milner J. D., Kastner D. L., Aksentijevich I., A hypermorphic missense mutation in PLCG2, encoding phospholipase Cγ2, causes a dominantly inherited autoinflammatory disease with immunodeficiency. Am. J. Hum. Genet. 91, 713–720 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Neves J. F., Doffinger R., Barcena-Morales G., Martins C., Papapietro O., Plagnol V., Curtis J., Martins M., Kumararatne D., Cordeiro A. I., Neves C., Borrego L. M., Katan M., Nejentsev S., Novel PLCG2 mutation in a patient with APLAID and cutis laxa. Front. Immunol. 9, 2863 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Martín-Nalda A., Fortuny C., Rey L., Bunney T. D., Alsina L., Esteve-Solé A., Bull D., Anton M. C., Basagaña M., Casals F., Deyá A., García-Prat M., Gimeno R., Juan M., Martinez-Banaclocha H., Martinez-Garcia J. J., Mensa-Vilaró A., Rabionet R., Martin-Begue N., Rudilla F., Yagüe J., Estivill X., García-Patos V., Pujol R. M., Soler-Palacín P., Katan M., Pelegrín P., Colobran R., Vicente A., Arostegui J. I., Severe autoinflammatory manifestations and antibody deficiency due to novel hypermorphic PLCG2 mutations. J. Clin. Immunol. 40, 987–1000 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Novice T., Kariminia A., Del Bel K. L., Lu H., Sharma M., Lim C. J., Read J., Vander Lugt M., Hannibal M. C., O’Dwyer D., Hosler M., Scharnitz T., Rizzo J. M., Zacur J., Priatel J., Abdossamadi S., Bohm A., Junker A., Turvey S. E., Schultz K. R., Rozmus J., A germline mutation in the C2 domain of PLCγ2 associated with gain-of-function expands the phenotype for PLCG2-related diseases. J. Clin. Immunol. 40, 267–276 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Ombrello M. J., Remmers E. F., Sun G., Freeman A. F., Datta S., Torabi-Parizi P., Subramanian N., Bunney T. D., Baxendale R. W., Martins M. S., Romberg N., Komarow H., Aksentijevich I., Kim H. S., Ho J., Cruse G., Jung M.-Y., Gilfillan A. M., Metcalfe D. D., Nelson C., O’Brien M., Wisch L., Stone K., Douek D. C., Gandhi C., Wanderer A. A., Lee H., Nelson S. F., Shianna K. V., Cirulli E. T., Goldstein D. B., Long E. O., Moir S., Meffre E., Holland S. M., Kastner D. L., Katan M., Hoffman H. M., Milner J. D., Cold urticaria, immunodeficiency, and autoimmunity related to PLCG2 deletions. N. Engl. J. Med. 366, 330–338 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Yu P., Constien R., Dear N., Katan M., Hanke P., Bunney T. D., Kunder S., Quintanilla-Martinez L., Huffstadt U., Schröder A., Jones N. P., Peters T., Fuchs H., Hrabe De Angelis M., Nehls M., Grosse J., Wabnitz P., Meyer T. P. H., Yasuda K., Schiemann M., Schneider-Fresenius C., Jagla W., Russ A., Popp A., Josephs M., Marquardt A., Laufs J., Schmittwolf C., Wagner H., Pfeffer K., Mudde G. C., Autoimmunity and inflammation due to a gain-of-function mutation in phospholipase Cγ2 that specifically increases external Ca²⁺ entry. Immunity 22, 451–465 (2005). [DOI] [PubMed] [Google Scholar]
9.Abe K., Fuchs H., Boersma A., Hans W., Yu P., Kalaydjiev S., Klaften M., Adler T., Calzada-Wack J., Mossbrugger I., Rathkolb B., Rozman J., Prehn C., Maraslioglu M., Kametani Y., Shimada S., Adamski J., Busch D. H., Esposito I., Klingenspor M., Wolf E., Wurst W., Gailus-Durner V., Katan M., Marschall S., Soewarto D., Wagner S., De Angelis M. H., A novel N-ethyl-N-nitrosourea-induced mutation in phospholipase Cγ2 causes inflammatory arthritis, metabolic defects, and male infertility in vitro in a murine model. Arthritis Rheum. 63, 1301–1311 (2011). [DOI] [PubMed] [Google Scholar]
10.Jones D., Woyach J. A., Zhao W., Caruthers S., Tu H., Coleman J., Byrd J. C., Johnson A. J., Lozanski G., PLCG2 C2 domain mutations co-occur with BTK and PLCG2 resistance mutations in chronic lymphocytic leukemia undergoing ibrutinib treatment. Leukemia 31, 1645–1647 (2017). [DOI] [PubMed] [Google Scholar]
11.Sedlarikova L., Petrackova A., Papajik T., Turcsanyi P., Kriegova E., Resistance-associated mutations in chronic lymphocytic leukemia patients treated with novel agents. Front. Oncol. 10, 894 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Sims R., Van Der Lee S. J., Naj A. C., Bellenguez C., Badarinarayan N., Jakobsdottir J., Kunkle B. W., Boland A., Raybould R., Bis J. C., Martin E. R., Grenier-Boley B., Heilmann-Heimbach S., Chouraki V., Kuzma A. B., Sleegers K., Vronskaya M., Ruiz A., Graham R. R., Olaso R., Hoffmann P., Grove M. L., Vardarajan B. N., Hiltunen M., Nöthen M. M., White C. C., Hamilton-Nelson K. L., Epelbaum J., Maier W., Choi S. H., Beecham G. W., Dulary C., Herms S., Smith A. V., Funk C. C., Derbois C., Forstner A. J., Ahmad S., Li H., Bacq D., Harold D., Satizabal C. L., Valladares O., Squassina A., Thomas R., Brody J. A., Qu L., Sánchez-Juan P., Morgan T., Wolters F. J., Zhao Y., Garcia F. S., Denning N., Fornage M., Malamon J., Naranjo M. C. D., Majounie E., Mosley T. H., Dombroski B., Wallon D., Lupton M. K., Dupuis J., Whitehead P., Fratiglioni L., Medway C., Jian X., Mukherjee S., Keller L., Brown K., Lin H., Cantwell L. B., Panza F., McGuinness B., Moreno-Grau S., Burgess J. D., Solfrizzi V., Proitsi P., Adams H. H., Allen M., Seripa D., Pastor P., Cupples L. A., Price N. D., Hannequin D., Frank-García A., Levy D., Chakrabarty P., Caffarra P., Giegling I., Beiser A. S., Giedraitis V., Hampel H., Garcia M. E., Wang X., Lannfelt L., Mecocci P., Eiriksdottir G., Crane P. K., Pasquier F., Boccardi V., Henández I., Barber R. C., Scherer M., Tarraga L., Adams P. M., Leber M., Chen Y., Albert M. S., Riedel-Heller S., Emilsson V., Beekly D., Braae A., Schmidt R., Blacker D., Masullo C., Schmidt H., Doody R. S., Spalletta G., Jr W. T. L., Fairchild T. J., Bossù P., Lopez O. L., Frosch M. P., Sacchinelli E., Ghetti B., Yang Q., Huebinger R. M., Jessen F., Li S., Kamboh M. I., Morris J. C., Sotolongo-Grau O., Katz M. J., Corcoran C., Dunstan M., Braddel A., Thomas C., Meggy A., Marshall R., Gerrish A., Chapman J., Aguilar M., Taylor S., Hill M., Fairén M. D., Hodges A., Vellas B., Soininen H., Kloszewska I., Daniilidou M., Uphill J., Patel Y., Hughes J. T., Lord J., Turton J., Hartmann A. M., Cecchetti R., Fenoglio C., Serpente M., Arcaro M., Caltagirone C., Orfei M. D., Ciaramella A., Pichler S., Mayhaus M., Gu W., Lleó A., Fortea J., Blesa R., Barber I. S., Brookes K., Cupidi C., Maletta R. G., Carrell D., Sorbi S., Moebus S., Urbano M., Pilotto A., Kornhuber J., Bosco P., Todd S., Craig D., Johnston J., Gill M., Lawlor B., Lynch A., Fox N. C., Hardy J., Albin R. L., Apostolova L. G., Arnold S. E., Asthana S., Atwood C. S., Baldwin C. T., Barnes L. L., Barral S., Beach T. G., Becker J. T., Bigio E. H., Bird T. D., Boeve B. F., Bowen J. D., Boxer A., Burke J. R., Burns J. M., Buxbaum J. D., Cairns N. J., Cao C., Carlson C. S., Carlsson C. M., Carney R. M., Carrasquillo M. M., Carroll S. L., Diaz C. C., Chui H. C., Clark D. G., Cribbs D. H., Crocco E. A., Decarli C., Dick M., Duara R., Evans D. A., Faber K. M., Fallon K. B., Fardo D. W., Farlow M. R., Ferris S., Foroud T. M., Galasko D. R., Gearing M., Geschwind D. H., Gilbert J. R., Graff-Radford N. R., Green R. C., Growdon J. H., Hamilton R. L., Harrell L. E., Honig L. S., Huentelman M. J., Hulette C. M., Hyman B. T., Jarvik G. P., Abner E., Jin L. W., Jun G., Karydas A., Kaye J. A., Kim R., Kowall N. W., Kramer J. H., Laferla F. M., Lah J. J., Leverenz J. B., Levey A. I., Li G., Lieberman A. P., Lunetta K. L., Lyketsos C. G., Marson D. C., Martiniuk F., Mash D. C., Masliah E., McCormick W. C., McCurry S. M., McDavid A. N., McKee A. C., Mesulam M., Miller B. L., Miller C. A., Miller J. W., Murrell J. R., Myers A. J., O’Bryant S., Olichney J. M., Pankratz V. S., Parisi J. E., Paulson H. L., Perry W., Peskind E., Pierce A., Poon W. W., Potter H., Quinn J. F., Raj A., Raskind M., Reisberg B., Reitz C., Ringman J. M., Roberson E. D., Rogaeva E., Rosen H. J., Rosenberg R. N., Sager M. A., Saykin A. J., Schneider J. A., Schneider L. S., Seeley W. W., Smith A. G., Sonnen J. A., Spina S., Stern R. A., Swerdlow R. H., Tanzi R. E., Thornton-Wells T. A., Trojanowski J. Q., Troncoso J. C., Van Deerlin V. M., Van Eldik L. J., Vinters H. V., Vonsattel J. P., Weintraub S., Welsh-Bohmer K. A., Wilhelmsen K. C., Williamson J., Wingo T. S., Woltjer R. L., Wright C. B., Yu C. E., Yu L., Garzia F., Golamaully F., Septier G., Engelborghs S., Vandenberghe R., De Deyn P. P., Fernadez C. M., Benito Y. A., Thonberg H., Forsell C., Lilius L., Kinhult-Stählbom A., Kilander L., Brundin R., Concari L., Helisalmi S., Koivisto A. M., Haapasalo A., Dermecourt V., Fievet N., Hanon O., Dufouil C., Brice A., Ritchie K., Dubois B., Himali J. J., Keene C. D., Tschanz J., Fitzpatrick A. L., Kukull W. A., Norton M., Aspelund T., Larson E. B., Munger R., Rotter J. I., Lipton R. B., Bullido M. J., Hofman A., Montine T. J., Coto E., Boerwinkle E., Petersen R. C., Alvarez V., Rivadeneira F., Reiman E. M., Gallo M., O’Donnell C. J., Reisch J. S., Bruni A. C., Royall D. R., Dichgans M., Sano M., Galimberti D., George-Hyslop P. S., Scarpini E., Tsuang D. W., Mancuso M., Bonuccelli U., Winslow A. R., Daniele A., Wu C. K., Peters O., Nacmias B., Riemenschneider M., Heun R., Brayne C., Rubinsztein D. C., Bras J., Guerreiro R., Al-Chalabi A., Shaw C. E., Collinge J., Tsolaki M., Clarimón J., Sussams R., Lovestone S., O’Donovan M. C., Owen M. J., Behrens T. W., Mead S., Uitterlinden A. G., Holmes C., Cruchaga C., Ingelsson M., Bennett D. A., Powell J., Golde T. E., Graff C., De Jager P. L., Morgan K., Ertekin-Taner N., Combarros O., Psaty B. M., Passmore P., Younkin S. G., Berr C., Gudnason V., Rujescu D., Dickson D. W., Dartigues J. F., Destefano A. L., Ortega-Cubero S., Hakonarson H., Campion D., Boada M., Kauwe J. K., Farrer L. A., Van Broeckhoven C., Ikram M. A., Jones L., Haines J. L., Tzourio C., Tzourio C., Escott-Price V., Mayeux R., Deleuze J. F., Amin N., Goate A. M., Pericak-Vance M. A., Amouyel P., Van Duijn C. M., Ramirez A., Wang L. S., Lambert J. C., Seshadri S., Williams J., Schellenberg G. D., Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer’s disease. Nat. Genet. 49, 1373–1384 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Conway O. J., Carrasquillo M. M., Wang X., Bredenberg J. M., Reddy J. S., Strickland S. L., Younkin C. S., Burgess J. D., Allen M., Lincoln S. J., Nguyen T., Malphrus K. G., Soto A. I., Walton R. L., Boeve B. F., Petersen R. C., Lucas J. A., Ferman T. J., Cheshire W. P., van Gerpen J. A., Uitti R. J., Wszolek Z. K., Ross O. A., Dickson D. W., Graff-Radford N. R., Ertekin-Taner N., ABI3 and PLCG2 missense variants as risk factors for neurodegenerative diseases in Caucasians and African Americans. Mol. Neurodegener. 13, 53 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Tesi N., van der Lee S. J., Hulsman M., Jansen I. E., Stringa N., van Schoor N., Meijers-Heijboer H., Huisman M., Scheltens P., Reinders M. J. T., van der Flier W. M., Holstege H., Centenarian controls increase variant effect sizes by an average twofold in an extreme case-extreme control analysis of Alzheimer’s disease. Eur. J. Hum. Genet. 27, 244–253 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Dalmasso M. C., Brusco L. I., Olivar N., Muchnik C., Hanses C., Milz E., Becker J., Heilmann-Heimbach S., Hoffmann P., Prestia F. A., Galeano P., Avalos M. S. S., Martinez L. E., Carulla M. E., Azurmendi P. J., Liberczuk C., Fezza C., Sampaño M., Fierens M., Jemar G., Solis P., Medel N., Lisso J., Sevillano Z., Bosco P., Bossù P., Spalletta G., Galimberti D., Mancuso M., Nacmias B., Sorbi S., Mecocci P., Pilotto A., Caffarra P., Panza F., Bullido M., Clarimon J., Sánchez-Juan P., Coto E., Sanchez-Garcia F., Graff C., Ingelsson M., Bellenguez C., Castaño E. M., Kairiyama C., Politis D. G., Kochen S., Scaro H., Maier W., Jessen F., Mangone C. A., Lambert J. C., Morelli L., Ramirez A., Transethnic meta-analysis of rare coding variants in PLCG2, ABI3, and TREM2 supports their general contribution to Alzheimer’s disease. Transl. Psychiatry 9, 55 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
16.van der Lee S. J., Conway O. J., Jansen I., Carrasquillo M. M., Kleineidam L., van den Akker E., Hernández I., van Eijk K. R., Stringa N., Chen J. A., Zettergren A., Andlauer T. F. M., Diez-Fairen M., Simon-Sanchez J., Lleó A., Zetterberg H., Nygaard M., Blauwendraat C., Savage J. E., Mengel-From J., Moreno-Grau S., Wagner M., Fortea J., Keogh M. J., Blennow K., Skoog I., Friese M. A., Pletnikova O., Zulaica M., Lage C., de Rojas I., Riedel-Heller S., Illán-Gala I., Wei W., Jeune B., Orellana A., Bergh F. T., Wang X., Hulsman M., Beker N., Tesi N., Morris C. M., Indakoetxea B., Collij L. E., Scherer M., Morenas-Rodríguez E., Ironside J. W., van Berckel B. N. M., Alcolea D., Wiendl H., Strickland S. L., Pastor P., Rodríguez E. R., Boeve B. F., Petersen R. C., Ferman T. J., van Gerpen J. A., Reinders M. J. T., Uitti R. J., Tárraga L., Maier W., Dols-Icardo O., Kawalia A., Dalmasso M. C., Boada M., Zettl U. K., van Schoor N. M., Beekman M., Allen M., Masliah E., de Munain A. L., Pantelyat A., Wszolek Z. K., Ross O. A., Dickson D. W., Graff-Radford N. R., Knopman D., Rademakers R., Lemstra A. W., Pijnenburg Y. A. L., Scheltens P., Gasser T., Chinnery P. F., Hemmer B., Huisman M. A., Troncoso J., Moreno F., Nohr E. A., Sørensen T. I. A., Heutink P., Sánchez-Juan P., Posthuma D., Coppola G., Karydas A. M., Varpetian A., Foroud T. M., Levey A. I., Kukull W. A., Mendez M. F., Ringman J., Chui H., Cotman C., DeCarli C., Miller B. L., Geschwind D. H., Clarimón J., Christensen K., Ertekin-Taner N., Scholz S. W., Ramirez A., Ruiz A., Slagboom E., van der Flier W. M., Holstege H., A nonsynonymous mutation in PLCG2 reduces the risk of Alzheimer’s disease, dementia with Lewy bodies and frontotemporal dementia, and increases the likelihood of longevity. Acta Neuropathol. 138, 237–250 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
17.van der Lee S. J., Conway O. J., Jansen I., Carrasquillo M. M., Kleineidam L., van den Akker E., Hernández I., van Eijk K. R., Stringa N., Chen J. A., Zettergren A., Andlauer T. F. M., Diez-Fairen M., Simon-Sanchez J., Lleó A., Zetterberg H., Nygaard M., Blauwendraat C., Savage J. E., Mengel-From J., Moreno-Grau S., Wagner M., Fortea J., Keogh M. J., Blennow K., Skoog I., Friese M. A., Pletnikova O., Zulaica M., Lage C., de Rojas I., Riedel-Heller S., Illán-Gala I., Wei W., Jeune B., Orellana A., Then Bergh F., Wang X., Hulsman M., Beker N., Tesi N., Morris C. M., Indakoetxea B., Collij L. E., Scherer M., Morenas-Rodríguez E., Ironside J. W., van Berckel B. N. M., Alcolea D., Wiendl H., Strickland S. L., Pastor P., Rodríguez Rodríguez E., Mead S., Synofzik M., van Swieten J. C., Leber I., Ferrari R., Hernandez D. G., Nalls M. A., Rohrer J. D., Ramasamy A., Kwok J. B. J., Dobson-Stone C., Schofield P. R., Halliday G. M., Hodges J. R., Piguet O., Bartley L., Thompson E., Borroni B., Padovani A., Cruchaga C., Cairns N. J., Benussi L., Binetti G., Ghidoni R., Forloni G., Albani D., Galimberti D., Fenoglio C., Serpente M., Scarpini E., Blesa R., Landqvist Waldö M., Nilsson K., Nilsson C., Mackenzie I. R. A., Hsiung G. Y. R., Mann D. M. A., Grafman J., Morris C. M., Attems J., Griffiths T. D., McKeith I. G., Thomas A. J., Pietrini P., Huey E. D., Wassermann E. M., Baborie A., Jaros E., Tierney M. C., Razquin C., Ortega-Cubero S., Alonso E., Perneczky R., Diehl-Schmid J., Alexopoulos P., Kurz A., Rainero I., Rubino E., Pinessi L., Rogaeva E., St George-Hyslop P., Rossi G., Tagliavini F., Giaccone G., Rowe J. B., Schlachetzki J. C. M., Uphill J., Collinge J., Danek A., Van Deerlin V. M., Grossman M., Trojanowski J. Q., van der Zee J., Van Broeckhoven C., Cappa S. F., Hannequin D., Golfier V., Vercelletto M., Brice A., Nacmias B., Sorbi S., Bagnoli S., Piaceri I., Nielsen J. E., Hjermind L. E., Riemenschneider M., Mayhaus M., Ibach B., Gasparoni G., Pichler S., Gu W., Rossor M. N., Fox N. C., Warren J. D., Spillantini M. G., Morris H. R., Rizzu P., Snowden J. S., Rollinson S., Richardson A., Gerhard A., Bruni A. C., Maletta R., Frangipane F., Cupidi C., Bernardi L., Anfossi M., Gallo M., Conidi M. E., Smirne N., Baker M., Josephs K. A., Parisi J. E., Seeley W. W., Miller B. L., Karydas A. M., Rosen H., Dopper E. G. P., Seelaar H., Logroscino G., Capozzo R., Novelli V., Puca A. A., Franceschi M., Postiglione A., Milan G., Sorrentino P., Kristiansen M., Chiang H. H., Graff C., Pasquier F., Rollin A., Deramecourt V., Lebouvier T., Kapogiannis D., Ferrucci L., Pickering-Brown S., Singleton A. B., Hardy J., Momeni P., Boeve B. F., Petersen R. C., Ferman T. J., van Gerpen J. A., Reinders M. J. T., Uitti R. J., Tárraga L., Maier W., Dols-Icardo O., Kawalia A., Dalmasso M. C., Boada M., Zettl U. K., van Schoor N. M., Beekman M., Allen M., Masliah E., de Munain A. L., Pantelyat A., Wszolek Z. K., Ross O. A., Dickson D. W., Graff-Radford N. R., Knopman D., Rademakers R., Lemstra A. W., Pijnenburg Y. A. L., Scheltens P., Gasser T., Chinnery P. F., Hemmer B., Huisman M. A., Troncoso J., Moreno F., Nohr E. A., Sørensen T. I. A., Heutink P., Sánchez-Juan P., Posthuma D., Coppola G., Karydas A. M., Varpetian A., Foroud T. M., Levey A. I., Kukull W. A., Mendez M. F., Ringman J., Chui H., Cotman C., DeCarli C., Geschwind D. H., Clarimón J., Christensen K., Ertekin-Taner N., Scholz S. W., Ramirez A., Ruiz A., Slagboom E., van der Flier W. M., Holstege H., Correction to: A nonsynonymous mutation in PLCG2 reduces the risk of Alzheimer's disease, dementia with Lewy bodies and frontotemporal dementia, and increases the likelihood of longevity. Acta Neuropathol. 139, 959–962 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Li K., Ran B., Wang Y., Liu L., Li W., PLCγ2 impacts microglia-related effectors revealing variants and pathways important in Alzheimer’s disease. Front. Cell Dev. Biol. 10, 1–13 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Kunkle B. W., Grenier-Boley B., Sims R., Bis J. C., Damotte V., Naj A. C., Boland A., Vronskaya M., van der Lee S. J., Amlie-Wolf A., Bellenguez C., Frizatti A., Chouraki V., Martin E. R., Sleegers K., Badarinarayan N., Jakobsdottir J., Hamilton-Nelson K. L., Moreno-Grau S., Olaso R., Raybould R., Chen Y., Kuzma A. B., Hiltunen M., Morgan T., Ahmad S., Vardarajan B. N., Epelbaum J., Hoffmann P., Boada M., Beecham G. W., Garnier J. G., Harold D., Fitzpatrick A. L., Valladares O., Moutet M. L., Gerrish A., Smith A. V., Qu L., Bacq D., Denning N., Jian X., Zhao Y., Del Zompo M., Fox N. C., Choi S. H., Mateo I., Hughes J. T., Adams H. H., Malamon J., Sanchez-Garcia F., Patel Y., Brody J. A., Dombroski B. A., Naranjo M. C. D., Daniilidou M., Eiriksdottir G., Mukherjee S., Wallon D., Uphill J., Aspelund T., Cantwell L. B., Garzia F., Galimberti D., Hofer E., Butkiewicz M., Fin B., Scarpini E., Sarnowski C., Bush W. S., Meslage S., Kornhuber J., White C. C., Song Y., Barber R. C., Engelborghs S., Sordon S., Voijnovic D., Adams P. M., Vandenberghe R., Mayhaus M., Cupples L. A., Albert M. S., De Deyn P. P., Gu W., Himali J. J., Beekly D., Squassina A., Hartmann A. M., Orellana A., Blacker D., Rodriguez-Rodriguez E., Lovestone S., Garcia M. E., Doody R. S., Munoz-Fernadez C., Sussams R., Lin H., Fairchild T. J., Benito Y. A., Holmes C., Karamujić-Čomić H., Frosch M. P., Thonberg H., Maier W., Roschupkin G., Ghetti B., Giedraitis V., Kawalia A., Li S., Huebinger R. M., Kilander L., Moebus S., Hernández I., Kamboh M. I., Brundin R. M., Turton J., Yang Q., Katz M. J., Concari L., Lord J., Beiser A. S., Keene C. D., Helisalmi S., Kloszewska I., Kukull W. A., Koivisto A. M., Lynch A., Tarraga L., Larson E. B., Haapasalo A., Lawlor B., Mosley T. H., Lipton R. B., Solfrizzi V., Gill M., Longstreth W. T., Montine T. J., Frisardi V., Diez-Fairen M., Rivadeneira F., Petersen R. C., Deramecourt V., Alvarez I., Salani F., Ciaramella A., Boerwinkle E., Reiman E. M., Fievet N., Rotter J. I., Reisch J. S., Hanon O., Cupidi C., Uitterlinden A. G. A., Royall D. R., Dufouil C., Maletta R. G., de Rojas I., Sano M., Brice A., Cecchetti R., George-Hyslop P. S., Ritchie K., Tsolaki M., Tsuang D. W., Dubois B., Craig D., Wu C. K., Soininen H., Avramidou D., Albin R. L., Fratiglioni L., Germanou A., Apostolova L. G., Keller L., Koutroumani M., Arnold S. E., Panza F., Gkatzima O., Asthana S., Hannequin D., Whitehead P., Atwood C. S., Caffarra P., Hampel H., Quintela I., Carracedo Á., Lannfelt L., Rubinsztein D. C., Barnes L. L., Pasquier F., Frölich L., Barral S., McGuinness B., Beach T. G., Johnston J. A., Becker J. T., Passmore P., Bigio E. H., Schott J. M., Bird T. D., Warren J. D., Boeve B. F., Lupton M. K., Bowen J. D., Proitsi P., Boxer A., Powell J. F., Burke J. R., Kauwe J. S. K., Burns J. M., Mancuso M., Buxbaum J. D., Bonuccelli U., Cairns N. J., McQuillin A., Cao C., Livingston G., Carlson C. S., Bass N. J., Carlsson C. M., Hardy J., Carney R. M., Bras J., Carrasquillo M. M., Guerreiro R., Allen M., Chui H. C., Fisher E., Masullo C., Crocco E. A., DeCarli C., Bisceglio G., Dick M., Ma L., Duara R., Graff-Radford N. R., Evans D. A., Hodges A., Faber K. M., Scherer M., Fallon K. B., Riemenschneider M., Fardo D. W., Heun R., Farlow M. R., Kölsch H., Ferris S., Leber M., Foroud T. M., Heuser I., Galasko D. R., Giegling I., Gearing M., Hüll M., Geschwind D. H., Gilbert J. R., Morris J. C., Green R. C., Mayo K., Growdon J. H., Feulner T., Hamilton R. L., Harrell L. E., Drichel D., Honig L. S., Cushion T. D., Huentelman M. J., Hollingworth P., Hulette C. M., Hyman B. T., Marshall R., Jarvik G. P., Meggy A., Abner E., Menzies G. E., Jin L. W., Leonenko G., Real L. M., Jun G. R., Baldwin C. T., Grozeva D., Karydas A., Russo G., Kaye J. A., Kim R., Jessen F., Kowall N. W., Vellas B., Kramer J. H., Vardy E., LaFerla F. M., Jöckel K. H., Lah J. J., Dichgans M., Leverenz J. B., Mann D., Levey A. I., Pickering-Brown S., Lieberman A. P., Klopp N., Lunetta K. L., Wichmann H. E., Lyketsos C. G., Morgan K., Marson D. C., Brown K., Martiniuk F., Medway C., Mash D. C., Nöthen M. M., Masliah E., Hooper N. M., McCormick W. C., Daniele A., McCurry S. M., Bayer A., McDavid A. N., Gallacher J., McKee A. C., van den Bussche H., Mesulam M., Brayne C., Miller B. L., Riedel-Heller S., Miller C. A., Miller J. W., Al-Chalabi A., Shaw C. E., Myers A. J., Wiltfang J., O’Bryant S., Olichney J. M., Alvarez V., Parisi J. E., Singleton A. B., Paulson H. L., Collinge J., Perry W. R., Mead S., Peskind E., Cribbs D. H., Rossor M., Pierce A., Ryan N. S., Poon W. W., Nacmias B., Potter H., Sorbi S., Quinn J. F., Sacchinelli E., Raj A., Spalletta G., Raskind M., Caltagirone C., Bossù P., Orfei M. D., Reisberg B., Clarke R., Reitz C., Smith A. D., Ringman J. M., Warden D., Roberson E. D., Wilcock G., Rogaeva E., Bruni A. C., Rosen H. J., Gallo M., Rosenberg R. N., Ben-Shlomo Y., Sager M. A., Mecocci P., Saykin A. J., Pastor P., Cuccaro M. L., Vance J. M., Schneider J. A., Schneider L. S., Slifer S., Seeley W. W., Smith A. G., Sonnen J. A., Spina S., Stern R. A., Swerdlow R. H., Tang M., Tanzi R. E., Trojanowski J. Q., Troncoso J. C., Van Deerlin V. M., Van Eldik L. J., Vinters H. V., Vonsattel J. P., Weintraub S., Welsh-Bohmer K. A., Wilhelmsen K. C., Williamson J., Wingo T. S., Woltjer R. L., Wright C. B., Yu C. E., Yu L., Saba Y., Pilotto A., Bullido M. J., Peters O., Crane P. K., Bennett D., Bosco P., Coto E., Boccardi V., De Jager P. L., Lleo A., Warner N., Lopez O. L., Ingelsson M., Deloukas P., Cruchaga C., Graff C., Gwilliam R., Fornage M., Goate A. M., Sanchez-Juan P., Kehoe P. G., Amin N., Ertekin-Taner N., Berr C., Debette S., Love S., Launer L. J., Younkin S. G., Dartigues J. F., Corcoran C., Ikram M. A., Dickson D. W., Nicolas G., Campion D., Tschanz J. A., Schmidt H., Hakonarson H., Clarimon J., Munger R., Schmidt R., Farrer L. A., Van Broeckhoven C., O’Donovan M. C., DeStefano A. L., Jones L., Haines J. L., Deleuze J. F., Owen M. J., Gudnason V., Mayeux R., Escott-Price V., Psaty B. M., Ramirez A., Wang L. S., Ruiz A., van Duijn C. M., Holmans P. A., Seshadri S., Williams J., Amouyel P., Schellenberg G. D., Lambert J. C., Pericak-Vance M. A., Genetic meta-analysis of diagnosed Alzheimer’s disease identifies new risk loci and implicates Aβ, tau, immunity and lipid processing. Nat. Genet. 51, 414–430 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Kunkle B. W., Grenier-Boley B., Sims R., Bis J. C., Damotte V., Naj A. C., Boland A., Vronskaya M., van der Lee S. J., Amlie-Wolf A., Bellenguez C., Frizatti A., Chouraki V., Martin E. R., Sleegers K., Badarinarayan N., Jakobsdottir J., Hamilton-Nelson K. L., Moreno-Grau S., Olaso R., Raybould R., Chen Y., Kuzma A. B., Hiltunen M., Morgan T., Ahmad S., Vardarajan B. N., Epelbaum J., Hoffmann P., Boada M., Beecham G. W., Garnier J. G., Harold D., Fitzpatrick A. L., Valladares O., Moutet M. L., Gerrish A., Smith A. V., Qu L., Bacq D., Denning N., Jian X., Zhao Y., Del Zompo M., Fox N. C., Choi S. H., Mateo I., Hughes J. T., Adams H. H., Malamon J., Sanchez-Garcia F., Patel Y., Brody J. A., Dombroski B. A., Naranjo M. C. D., Daniilidou M., Eiriksdottir G., Mukherjee S., Wallon D., Uphill J., Aspelund T., Cantwell L. B., Garzia F., Galimberti D., Hofer E., Butkiewicz M., Fin B., Scarpini E., Sarnowski C., Bush W. S., Meslage S., Kornhuber J., White C. C., Song Y., Barber R. C., Engelborghs S., Sordon S., Voijnovic D., Adams P. M., Vandenberghe R., Mayhaus M., Cupples L. A., Albert M. S., De Deyn P. P., Gu W., Himali J. J., Beekly D., Squassina A., Hartmann A. M., Orellana A., Blacker D., Rodriguez-Rodriguez E., Lovestone S., Garcia M. E., Doody R. S., Munoz-Fernadez C., Sussams R., Lin H., Fairchild T. J., Benito Y. A., Holmes C., Karamujić-Čomić H., Frosch M. P., Thonberg H., Maier W., Roshchupkin G., Ghetti B., Giedraitis V., Kawalia A., Li S., Huebinger R. M., Kilander L., Moebus S., Hernández I., Kamboh M. I., Brundin R. M., Turton J., Yang Q., Katz M. J., Concari L., Lord J., Beiser A. S., Keene C. D., Helisalmi S., Kloszewska I., Kukull W. A., Koivisto A. M., Lynch A., Tarraga L., Larson E. B., Haapasalo A., Lawlor B., Mosley T. H., Lipton R. B., Solfrizzi V., Gill M., Longstreth W. T., Montine T. J., Frisardi V., Diez-Fairen M., Rivadeneira F., Petersen R. C., Deramecourt V., Alvarez I., Salani F., Ciaramella A., Boerwinkle E., Reiman E. M., Fievet N., Rotter J. I., Reisch J. S., Hanon O., Cupidi C., Uitterlinden A. G. A., Royall D. R., Dufouil C., Maletta R. G., de Rojas I., Sano M., Brice A., Cecchetti R., George-Hyslop P. S., Ritchie K., Tsolaki M., Tsuang D. W., Dubois B., Craig D., Wu C. K., Soininen H., Avramidou D., Albin R. L., Fratiglioni L., Germanou A., Apostolova L. G., Keller L., Koutroumani M., Arnold S. E., Panza F., Gkatzima O., Asthana S., Hannequin D., Whitehead P., Atwood C. S., Caffarra P., Hampel H., Quintela I., Carracedo Á., Lannfelt L., Rubinsztein D. C., Barnes L. L., Pasquier F., Frölich L., Barral S., McGuinness B., Beach T. G., Johnston J. A., Becker J. T., Passmore P., Bigio E. H., Schott J. M., Bird T. D., Warren J. D., Boeve B. F., Lupton M. K., Bowen J. D., Proitsi P., Boxer A., Powell J. F., Burke J. R., Kauwe J. S. K., Burns J. M., Mancuso M., Buxbaum J. D., Bonuccelli U., Cairns N. J., McQuillin A., Cao C., Livingston G., Carlson C. S., Bass N. J., Carlsson C. M., Hardy J., Carney R. M., Bras J., Carrasquillo M. M., Guerreiro R., Allen M., Chui H. C., Fisher E., Masullo C., Crocco E. A., DeCarli C., Bisceglio G., Dick M., Ma L., Duara R., Graff-Radford N. R., Evans D. A., Hodges A., Faber K. M., Scherer M., Fallon K. B., Riemenschneider M., Fardo D. W., Heun R., Farlow M. R., Kölsch H., Ferris S., Leber M., Foroud T. M., Heuser I., Galasko D. R., Giegling I., Gearing M., Hüll M., Geschwind D. H., Gilbert J. R., Morris J. C., Green R. C., Mayo K., Growdon J. H., Feulner T., Hamilton R. L., Harrell L. E., Drichel D., Honig L. S., Cushion T. D., Huentelman M. J., Hollingworth P., Hulette C. M., Hyman B. T., Marshall R., Jarvik G. P., Meggy A., Abner E., Menzies G. E., Jin L. W., Leonenko G., Real L. M., Jun G. R., Baldwin C. T., Grozeva D., Karydas A., Russo G., Kaye J. A., Kim R., Jessen F., Kowall N. W., Vellas B., Kramer J. H., Vardy E., LaFerla F. M., Jöckel K. H., Lah J. J., Dichgans M., Leverenz J. B., Mann D., Levey A. I., Pickering-Brown S., Lieberman A. P., Klopp N., Lunetta K. L., Wichmann H. E., Lyketsos C. G., Morgan K., Marson D. C., Brown K., Martiniuk F., Medway C., Mash D. C., Nöthen M. M., Masliah E., Hooper N. M., McCormick W. C., Daniele A., McCurry S. M., Bayer A., McDavid A. N., Gallacher J., McKee A. C., van den Bussche H., Mesulam M., Brayne C., Miller B. L., Riedel-Heller S., Miller C. A., Miller J. W., Al-Chalabi A., Shaw C. E., Myers A. J., Wiltfang J., O’Bryant S., Olichney J. M., Alvarez V., Parisi J. E., Singleton A. B., Paulson H. L., Collinge J., Perry W. R., Mead S., Peskind E., Cribbs D. H., Rossor M., Pierce A., Ryan N. S., Poon W. W., Nacmias B., Potter H., Sorbi S., Quinn J. F., Sacchinelli E., Raj A., Spalletta G., Raskind M., Caltagirone C., Bossù P., Orfei M. D., Reisberg B., Clarke R., Reitz C., Smith A. D., Ringman J. M., Warden D., Roberson E. D., Wilcock G., Rogaeva E., Bruni A. C., Rosen H. J., Gallo M., Rosenberg R. N., Ben-Shlomo Y., Sager M. A., Mecocci P., Saykin A. J., Pastor P., Cuccaro M. L., Vance J. M., Schneider J. A., Schneider L. S., Slifer S., Seeley W. W., Smith A. G., Sonnen J. A., Spina S., Stern R. A., Swerdlow R. H., Tang M., Tanzi R. E., Trojanowski J. Q., Troncoso J. C., Van Deerlin V. M., Van Eldik L. J., Vinters H. V., Vonsattel J. P., Weintraub S., Welsh-Bohmer K. A., Wilhelmsen K. C., Williamson J., Wingo T. S., Woltjer R. L., Wright C. B., Yu C. E., Yu L., Saba Y., Pilotto A., Bullido M. J., Peters O., Crane P. K., Bennett D., Bosco P., Coto E., Boccardi V., De Jager P. L., Lleo A., Warner N., Lopez O. L., Ingelsson M., Deloukas P., Cruchaga C., Graff C., Gwilliam R., Fornage M., Goate A. M., Sanchez-Juan P., Kehoe P. G., Amin N., Ertekin-Taner N., Berr C., Debette S., Love S., Launer L. J., Younkin S. G., Dartigues J. F., Corcoran C., Ikram M. A., Dickson D. W., Nicolas G., Campion D., Tschanz J. A., Schmidt H., Hakonarson H., Clarimon J., Munger R., Schmidt R., Farrer L. A., Van Broeckhoven C., O’Donovan M. C., DeStefano A. L., Jones L., Haines J. L., Deleuze J. F., Owen M. J., Gudnason V., Mayeux R., Escott-Price V., Psaty B. M., Ramirez A., Wang L. S., Ruiz A., van Duijn C. M., Holmans P. A., Seshadri S., Williams J., Amouyel P., Schellenberg G. D., Lambert J. C., Pericak-Vance M. A., Author correction: Genetic meta-analysis of diagnosed Alzheimer’s disease identifies new risk loci and implicates Aβ, tau, immunity and lipid processing (Nature Genetics, (2019), 51, 3, (414-430), 10.1038/s41588-019-0358-2). Nat. Genet. 51, 1423–1424 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Kleineidam L., Chouraki V., Próchnicki T., van der Lee S. J., Madrid-Márquez L., Wagner-Thelen H., Karaca I., Weinhold L., Wolfsgruber S., Boland A., Adami P. V. M., Lewczuk P., Popp J., Brosseron F., Jansen I. E., Hulsman M., Kornhuber J., Peters O., Berr C., Heun R., Frölich L., Tzourio C., Dartigues J. F., Hüll M., Espinosa A., Hernández I., de Rojas I., Orellana A., Valero S., Stringa N., van Schoor N. M., Huisman M., Scheltens P., Amouyel P., Ramirez A., Rüther E., Deleuze J. F., Wiltfang J., Tarraga L., Schmid M., Scherer M., Riedel-Heller S., Heneka M. T., Amouyel P., Jessen F., Boada M., Maier W., Schneider A., González-Pérez A., van der Flier W. M., Wagner M., Lambert J. C., Holstege H., Sáez M. E., Latz E., Ruiz A., PLCG2 protective variant p.P522R modulates tau pathology and disease progression in patients with mild cognitive impairment. Acta Neuropathol. 139, 1025–1044 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Magno L., Lessard C. B., Martins M., Lang V., Cruz P., Asi Y., Katan M., Bilsland J., Lashley T., Chakrabarty P., Golde T. E., Whiting P. J., Alzheimer’s disease phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph. Alzheimer’s Res. Ther. 11, 16 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Takalo M., Wittrahm R., Wefers B., Parhizkar S., Jokivarsi K., Kuulasmaa T., Mäkinen P., Martiskainen H., Wurst W., Xiang X., Marttinen M., Poutiainen P., Haapasalo A., Hiltunen M., Haass C., The Alzheimer’s disease-associated protective Plcγ2-P522R variant promotes immune functions. Mol. Neurodegener. 15, 1–14 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Maguire E., Menzies G. E., Phillips T., Sasner M., Williams H. M., Czubala M. A., Evans N., Cope E. L., Sims R., Howell G. R., Lloyd-Evans E., Williams J., Allen N. D., Taylor P. R., PIP2 depletion and altered endocytosis caused by expression of Alzheimer’s disease-protective variant PLCγ2 R522. EMBO J. 40, e105603 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Tsai A. P., Dong C., Lin P. B. C., Oblak A. L., Viana Di Prisco G., Wang N., Hajicek N., Carr A. J., Lendy E. K., Hahn O., Atkins M., Foltz A. G., Patel J., Xu G., Moutinho M., Sondek J., Zhang Q., Mesecar A. D., Liu Y., Atwood B. K., Wyss-Coray T., Nho K., Bissel S. J., Lamb B. T., Landreth G. E., Genetic variants of phospholipase C-γ2 alter the phenotype and function of microglia and confer differential risk for Alzheimer’s disease. Immunity 56, 2121–2136.e6 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Oblak A. L., Kotredes K. P., Pandey R. S., Reagan A. M., Ingraham C., Perkins B., Lloyd C., Baker D., Lin P. B., Soni D. M., Tsai A. P., Persohn S. A., Bedwell A. A., Eldridge K., Speedy R., Meyer J. A., Peters J. S., Figueiredo L. L., Sasner M., Territo P. R., Sukoff Rizzo S. J., Carter G. W., Lamb B. T., Howell G. R., Plcg2M28L interacts with high fat/high sugar diet to accelerate alzheimer’s disease-relevant phenotypes in mice. Front. Aging Neurosci. 14, 886575 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Hajicek N., Keith N. C., Siraliev-Perez E., Temple B. R. S., Huang W., Zhang Q., Harden T. K., Sondek J., Structural basis for the activation of plc-γ isozymes by phosphorylation and cancer-associated mutations. Elife 8, e51700 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Kim H. K., Kim J. W., Zilberstein A., Margolis B., Kim J. G., Schlessinger J., Rhee S. G., PDGF stimulation of inositol phospholipid hydrolysis requires PLC-γ1 phosphorylation on tyrosine residues 783 and 1254. Cell 65, 435–441 (1991). [DOI] [PubMed] [Google Scholar]
29.Gresset A., Hicks S. N., Harden T. K., Sondek J., Mechanism of phosphorylation-induced activation of phospholipase C-γ isozymes. J. Biol. Chem. 285, 35836–35847 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Liu Y., Bunney T. D., Khosa S., Macé K., Beckenbauer K., Askwith T., Maslen S., Stubbs C., de Oliveira T. M., Sader K., Skehel M., Gavin A. C., Phillips C., Katan M., Structural insights and activating mutations in diverse pathologies define mechanisms of deregulation for phospholipase C gamma enzymes. EBioMedicine 51, 102607 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Bae J. H., Lew E. D., Yuzawa S., Tomé F., Lax I., Schlessinger J., The selectivity of receptor tyrosine kinase signaling is controlled by a secondary SH2 domain binding site. Cell 138, 514–524 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Huang Z., Marsiglia W. M., Basu Roy U., Rahimi N., Ilghari D., Wang H., Chen H., Gai W., Blais S., Neubert T. A., Mansukhani A., Traaseth N. J., Li X., Mohammadi M., Two FGF receptor kinase molecules act in concert to recruit and transphosphorylate phospholipase Cγ. Mol. Cell 61, 98–110 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Peters K. G., Marie J., Wilson E., Ives H. E., Escobedo J., Del Rosario M., Mirda D., Williams L. T., Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca²⁺ flux but not mitogenesis. Nature 358, 678–681 (1992). [DOI] [PubMed] [Google Scholar]
34.Mohammadi M., Dionne C. A., Li W., Li N., Spivak T., Honegger A. M., Jaye M., Schlessinger J., Point mutat ion in FGF receptor eliminates phosphatidylinositol hydrolysis without affecting mitogenesis. Nature 358, 681–684 (1992). [DOI] [PubMed] [Google Scholar]
35.Watanabe D., Hashimoto S., Ishiai M., Matsushita M., Baba Y., Kishimoto T., Kurosaki T., Tsukada S., Four tyrosine residues in phospholipase C-γ2, identified as Btk-dependent phosphorylation sites, are required for B cell antigen receptor-coupled calcium signaling. J. Biol. Chem. 276, 38595–38601 (2001). [DOI] [PubMed] [Google Scholar]
36.Kim Y. J., Sekiya F., Poulin B., Bae Y. S., Rhee S. G., Mechanism of B-cell receptor-induced phosphorylation and activation of phospholipase C-γ2. Mol. Cell. Biol. 24, 9986–9999 (2004). [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Özdener F., Dangelmaier C., Ashby B., Kunapuli S. P., Daniel J. L., Activation of phospholipase Cγ2 by tyrosine phosphorylation. Mol. Pharmacol. 62, 672–679 (2002). [DOI] [PubMed] [Google Scholar]
38.Huang W., Carr A. J., Hajicek N., Sokolovski M., Siraliev-Perez E., Hardy P. B., Pearce K. H., Sondek J., Zhang Q., A high-throughput assay to identify allosteric inhibitors of the PLC-γ isozymes operating at membranes. Biochemistry 59, 4029–4038 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Jumper J., Evans R., Pritzel A., Green T., Figurnov M., Ronneberger O., Tunyasuvunakool K., Bates R., Žídek A., Potapenko A., Bridgland A., Meyer C., Kohl S. A. A., Ballard A. J., Cowie A., Romera-Paredes B., Nikolov S., Jain R., Adler J., Back T., Petersen S., Reiman D., Clancy E., Zielinski M., Steinegger M., Pacholska M., Berghammer T., Bodenstein S., Silver D., Vinyals O., Senior A. W., Kavukcuoglu K., Kohli P., Hassabis D., Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Varadi M., Anyango S., Deshpande M., Nair S., Natassia C., Yordanova G., Yuan D., Stroe O., Wood G., Laydon A., Zídek A., Green T., Tunyasuvunakool K., Petersen S., Jumper J., Clancy E., Green R., Vora A., Lutfi M., Figurnov M., Cowie A., Hobbs N., Kohli P., Kleywegt G., Birney E., Hassabis D., Velankar S., AlphaFold protein structure database: Massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 50, D439–D444 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Aslanidis C., de Jong P. J., Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18, 6069–6074 (1990). [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Siraliev-Perez E., Stariha J. T. B., Hoffmann R. M., Temple B. R. S., Zhang Q., Hajicek N., Jenkins M. L., Burke J. E., Sondek J., Dynamics of allosteric regulation of the phospholipase C-γ isozymes upon recruitment to membranes. Elife 11, e77809 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Gasteiger E., Gattiker A., Hoogland C., Ivanyi I., Appel R. D., Bairoch A., ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 31, 3784–3788 (2003). [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Wang Q. S., Yu F., Huang S., Sun B., Zhang K. H., Liu K., Wang Z. J., Xu C. Y., Wang S. S., Yang L. F., Pan Q. Y., Li L., Zhou H., Cui Y., Xu Q., Earnest T., He J. H., The macromolecular crystallography beamline of SSRF. Nucl. Sci. Tech. 26, 1–6 (2015). [Google Scholar]
45.Vonrhein C., Flensburg C., Keller P., Sharff A., Smart O., Paciorek W., Womack T., Bricogne G., Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. Sect. D Biol. Crystallogr. 67, 293–302 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Kabsch W., XDS. Acta Crystallogr D Biol Crystallogr 66, 125–132 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Winn M. D., Ballard C. C., Cowtan K. D., Dodson E. J., Emsley P., Evans P. R., Keegan R. M., Krissinel E. B., Leslie A. G. W., McCoy A., McNicholas S. J., Murshudov G. N., Pannu N. S., Potterton E. A., Powell H. R., Read R. J., Vagin A., Wilson K. S., Overview of the CCP4 suite and current developments. Acta Crystallogr. Sect. D Biol. Crystallogr. 67, 235–242 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
48.McCoy A. J., Grosse-Kunstleve R. W., Adams P. D., Winn M. D., Storoni L. C., Read R. J., Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007). [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Murshudov G. N., Skubák P., Lebedev A. A., Pannu N. S., Steiner R. A., Nicholls R. A., Winn M. D., Long F., Vagin A. A., REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. Sect. D Biol. Crystallogr. 67, 355–367 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Emsley P., Lohkamp B., Scott W. G., Cowtan K., Features and development of Coot. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 486–501 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
51.The PyMOL Molecular Graphics System, Version 2.3 Schrödinger LLC.
52.Zheng S. Q., Palovcak E., Armache J. P., Verba K. A., Cheng Y., Agard D. A., MotionCor2: Anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Rohou A., Grigorieff N., CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Ru H., Chambers M. G., Fu T. M., Tong A. B., Liao M., Wu H., Molecular mechanism of V(D)J recombination from synaptic RAG1-RAG2 complex structures. Cell 163, 1138–1152 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Zivanov J., Nakane T., Forsberg B. O., Kimanius D., Hagen W. J. H., Lindahl E., Scheres S. H. W., New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife 7, e42166 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Punjani A., Rubinstein J. L., Fleet D. J., Brubaker M. A., CryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017). [DOI] [PubMed] [Google Scholar]
57.Afonine P. V., Poon B. K., Read R. J., Sobolev O. V., Terwilliger T. C., Urzhumtsev A., Adams P. D., Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. Sect. D Struct. Biol. 74, 531–544 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
58.Pettersen E. F., Goddard T. D., Huang C. C., Meng E. C., Couch G. S., Croll T. I., Morris J. H., Ferrin T. E., ChimeraX U. C. S. F., Structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Emsley P., Cowtan K., Coot: Model-building tools for molecular graphics. Acta Crystallogr. Sect. D Biol. Crystallogr. 60, 2126–2132 (2004). [DOI] [PubMed] [Google Scholar]
60.Carr A. J., Siraliev-Perez E., Huang W., Sondek J., Zhang Q., Fluorogenic XY-69 in lipid vesicles for measuring activity of phospholipase C isozymes. Methods Mol. Biol. 2251, 225–236 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
61.Jing H., Reed A., Ulanovskaya O. A., Grigoleit J. S., Herbst D. M., Henry C. L., Li H., Barbas S., Germain J., Masuda K., Cravatt B. F., Phospholipase Cγ2 regulates endocannabinoid and eicosanoid networks in innate immune cells. Proc. Natl. Acad. Sci. U.S.A. 118, e2112971118 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figs. S1 to S8

Tables S1 and S2

sciadv.adn6037_sm.pdf^{(4.1MB, pdf)}

[R1] 1.Jackson J. T., Mulazzani E., Nutt S. L., Masters S. L., The role of PLCγ2 in immunological disorders, cancer, and neurodegeneration. J. Biol. Chem. 297, 100905 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R2] 2.Koss H., Bunney T. D., Behjati S., Katan M., Dysfunction of phospholipase Cγ in immune disorders and cancer. Trends Biochem. Sci. 39, 603–611 (2014). [DOI] [PubMed] [Google Scholar]

[R3] 3.Zhou Q., Lee G. S., Brady J., Datta S., Katan M., Sheikh A., Martins M. S., Bunney T. D., Santich B. H., Moir S., Kuhns D. B., Priel D. A. L., Ombrello A., Stone D., Ombrello M. J., Khan J., Milner J. D., Kastner D. L., Aksentijevich I., A hypermorphic missense mutation in PLCG2, encoding phospholipase Cγ2, causes a dominantly inherited autoinflammatory disease with immunodeficiency. Am. J. Hum. Genet. 91, 713–720 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R4] 4.Neves J. F., Doffinger R., Barcena-Morales G., Martins C., Papapietro O., Plagnol V., Curtis J., Martins M., Kumararatne D., Cordeiro A. I., Neves C., Borrego L. M., Katan M., Nejentsev S., Novel PLCG2 mutation in a patient with APLAID and cutis laxa. Front. Immunol. 9, 2863 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R5] 5.Martín-Nalda A., Fortuny C., Rey L., Bunney T. D., Alsina L., Esteve-Solé A., Bull D., Anton M. C., Basagaña M., Casals F., Deyá A., García-Prat M., Gimeno R., Juan M., Martinez-Banaclocha H., Martinez-Garcia J. J., Mensa-Vilaró A., Rabionet R., Martin-Begue N., Rudilla F., Yagüe J., Estivill X., García-Patos V., Pujol R. M., Soler-Palacín P., Katan M., Pelegrín P., Colobran R., Vicente A., Arostegui J. I., Severe autoinflammatory manifestations and antibody deficiency due to novel hypermorphic PLCG2 mutations. J. Clin. Immunol. 40, 987–1000 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R6] 6.Novice T., Kariminia A., Del Bel K. L., Lu H., Sharma M., Lim C. J., Read J., Vander Lugt M., Hannibal M. C., O’Dwyer D., Hosler M., Scharnitz T., Rizzo J. M., Zacur J., Priatel J., Abdossamadi S., Bohm A., Junker A., Turvey S. E., Schultz K. R., Rozmus J., A germline mutation in the C2 domain of PLCγ2 associated with gain-of-function expands the phenotype for PLCG2-related diseases. J. Clin. Immunol. 40, 267–276 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R7] 7.Ombrello M. J., Remmers E. F., Sun G., Freeman A. F., Datta S., Torabi-Parizi P., Subramanian N., Bunney T. D., Baxendale R. W., Martins M. S., Romberg N., Komarow H., Aksentijevich I., Kim H. S., Ho J., Cruse G., Jung M.-Y., Gilfillan A. M., Metcalfe D. D., Nelson C., O’Brien M., Wisch L., Stone K., Douek D. C., Gandhi C., Wanderer A. A., Lee H., Nelson S. F., Shianna K. V., Cirulli E. T., Goldstein D. B., Long E. O., Moir S., Meffre E., Holland S. M., Kastner D. L., Katan M., Hoffman H. M., Milner J. D., Cold urticaria, immunodeficiency, and autoimmunity related to PLCG2 deletions. N. Engl. J. Med. 366, 330–338 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R8] 8.Yu P., Constien R., Dear N., Katan M., Hanke P., Bunney T. D., Kunder S., Quintanilla-Martinez L., Huffstadt U., Schröder A., Jones N. P., Peters T., Fuchs H., Hrabe De Angelis M., Nehls M., Grosse J., Wabnitz P., Meyer T. P. H., Yasuda K., Schiemann M., Schneider-Fresenius C., Jagla W., Russ A., Popp A., Josephs M., Marquardt A., Laufs J., Schmittwolf C., Wagner H., Pfeffer K., Mudde G. C., Autoimmunity and inflammation due to a gain-of-function mutation in phospholipase Cγ2 that specifically increases external Ca²⁺ entry. Immunity 22, 451–465 (2005). [DOI] [PubMed] [Google Scholar]

[R9] 9.Abe K., Fuchs H., Boersma A., Hans W., Yu P., Kalaydjiev S., Klaften M., Adler T., Calzada-Wack J., Mossbrugger I., Rathkolb B., Rozman J., Prehn C., Maraslioglu M., Kametani Y., Shimada S., Adamski J., Busch D. H., Esposito I., Klingenspor M., Wolf E., Wurst W., Gailus-Durner V., Katan M., Marschall S., Soewarto D., Wagner S., De Angelis M. H., A novel N-ethyl-N-nitrosourea-induced mutation in phospholipase Cγ2 causes inflammatory arthritis, metabolic defects, and male infertility in vitro in a murine model. Arthritis Rheum. 63, 1301–1311 (2011). [DOI] [PubMed] [Google Scholar]

[R10] 10.Jones D., Woyach J. A., Zhao W., Caruthers S., Tu H., Coleman J., Byrd J. C., Johnson A. J., Lozanski G., PLCG2 C2 domain mutations co-occur with BTK and PLCG2 resistance mutations in chronic lymphocytic leukemia undergoing ibrutinib treatment. Leukemia 31, 1645–1647 (2017). [DOI] [PubMed] [Google Scholar]

[R11] 11.Sedlarikova L., Petrackova A., Papajik T., Turcsanyi P., Kriegova E., Resistance-associated mutations in chronic lymphocytic leukemia patients treated with novel agents. Front. Oncol. 10, 894 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R13] 13.Conway O. J., Carrasquillo M. M., Wang X., Bredenberg J. M., Reddy J. S., Strickland S. L., Younkin C. S., Burgess J. D., Allen M., Lincoln S. J., Nguyen T., Malphrus K. G., Soto A. I., Walton R. L., Boeve B. F., Petersen R. C., Lucas J. A., Ferman T. J., Cheshire W. P., van Gerpen J. A., Uitti R. J., Wszolek Z. K., Ross O. A., Dickson D. W., Graff-Radford N. R., Ertekin-Taner N., ABI3 and PLCG2 missense variants as risk factors for neurodegenerative diseases in Caucasians and African Americans. Mol. Neurodegener. 13, 53 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R14] 14.Tesi N., van der Lee S. J., Hulsman M., Jansen I. E., Stringa N., van Schoor N., Meijers-Heijboer H., Huisman M., Scheltens P., Reinders M. J. T., van der Flier W. M., Holstege H., Centenarian controls increase variant effect sizes by an average twofold in an extreme case-extreme control analysis of Alzheimer’s disease. Eur. J. Hum. Genet. 27, 244–253 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R15] 15.Dalmasso M. C., Brusco L. I., Olivar N., Muchnik C., Hanses C., Milz E., Becker J., Heilmann-Heimbach S., Hoffmann P., Prestia F. A., Galeano P., Avalos M. S. S., Martinez L. E., Carulla M. E., Azurmendi P. J., Liberczuk C., Fezza C., Sampaño M., Fierens M., Jemar G., Solis P., Medel N., Lisso J., Sevillano Z., Bosco P., Bossù P., Spalletta G., Galimberti D., Mancuso M., Nacmias B., Sorbi S., Mecocci P., Pilotto A., Caffarra P., Panza F., Bullido M., Clarimon J., Sánchez-Juan P., Coto E., Sanchez-Garcia F., Graff C., Ingelsson M., Bellenguez C., Castaño E. M., Kairiyama C., Politis D. G., Kochen S., Scaro H., Maier W., Jessen F., Mangone C. A., Lambert J. C., Morelli L., Ramirez A., Transethnic meta-analysis of rare coding variants in PLCG2, ABI3, and TREM2 supports their general contribution to Alzheimer’s disease. Transl. Psychiatry 9, 55 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R16] 16.van der Lee S. J., Conway O. J., Jansen I., Carrasquillo M. M., Kleineidam L., van den Akker E., Hernández I., van Eijk K. R., Stringa N., Chen J. A., Zettergren A., Andlauer T. F. M., Diez-Fairen M., Simon-Sanchez J., Lleó A., Zetterberg H., Nygaard M., Blauwendraat C., Savage J. E., Mengel-From J., Moreno-Grau S., Wagner M., Fortea J., Keogh M. J., Blennow K., Skoog I., Friese M. A., Pletnikova O., Zulaica M., Lage C., de Rojas I., Riedel-Heller S., Illán-Gala I., Wei W., Jeune B., Orellana A., Bergh F. T., Wang X., Hulsman M., Beker N., Tesi N., Morris C. M., Indakoetxea B., Collij L. E., Scherer M., Morenas-Rodríguez E., Ironside J. W., van Berckel B. N. M., Alcolea D., Wiendl H., Strickland S. L., Pastor P., Rodríguez E. R., Boeve B. F., Petersen R. C., Ferman T. J., van Gerpen J. A., Reinders M. J. T., Uitti R. J., Tárraga L., Maier W., Dols-Icardo O., Kawalia A., Dalmasso M. C., Boada M., Zettl U. K., van Schoor N. M., Beekman M., Allen M., Masliah E., de Munain A. L., Pantelyat A., Wszolek Z. K., Ross O. A., Dickson D. W., Graff-Radford N. R., Knopman D., Rademakers R., Lemstra A. W., Pijnenburg Y. A. L., Scheltens P., Gasser T., Chinnery P. F., Hemmer B., Huisman M. A., Troncoso J., Moreno F., Nohr E. A., Sørensen T. I. A., Heutink P., Sánchez-Juan P., Posthuma D., Coppola G., Karydas A. M., Varpetian A., Foroud T. M., Levey A. I., Kukull W. A., Mendez M. F., Ringman J., Chui H., Cotman C., DeCarli C., Miller B. L., Geschwind D. H., Clarimón J., Christensen K., Ertekin-Taner N., Scholz S. W., Ramirez A., Ruiz A., Slagboom E., van der Flier W. M., Holstege H., A nonsynonymous mutation in PLCG2 reduces the risk of Alzheimer’s disease, dementia with Lewy bodies and frontotemporal dementia, and increases the likelihood of longevity. Acta Neuropathol. 138, 237–250 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R18] 18.Li K., Ran B., Wang Y., Liu L., Li W., PLCγ2 impacts microglia-related effectors revealing variants and pathways important in Alzheimer’s disease. Front. Cell Dev. Biol. 10, 1–13 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R21] 21.Kleineidam L., Chouraki V., Próchnicki T., van der Lee S. J., Madrid-Márquez L., Wagner-Thelen H., Karaca I., Weinhold L., Wolfsgruber S., Boland A., Adami P. V. M., Lewczuk P., Popp J., Brosseron F., Jansen I. E., Hulsman M., Kornhuber J., Peters O., Berr C., Heun R., Frölich L., Tzourio C., Dartigues J. F., Hüll M., Espinosa A., Hernández I., de Rojas I., Orellana A., Valero S., Stringa N., van Schoor N. M., Huisman M., Scheltens P., Amouyel P., Ramirez A., Rüther E., Deleuze J. F., Wiltfang J., Tarraga L., Schmid M., Scherer M., Riedel-Heller S., Heneka M. T., Amouyel P., Jessen F., Boada M., Maier W., Schneider A., González-Pérez A., van der Flier W. M., Wagner M., Lambert J. C., Holstege H., Sáez M. E., Latz E., Ruiz A., PLCG2 protective variant p.P522R modulates tau pathology and disease progression in patients with mild cognitive impairment. Acta Neuropathol. 139, 1025–1044 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] 22.Magno L., Lessard C. B., Martins M., Lang V., Cruz P., Asi Y., Katan M., Bilsland J., Lashley T., Chakrabarty P., Golde T. E., Whiting P. J., Alzheimer’s disease phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph. Alzheimer’s Res. Ther. 11, 16 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R23] 23.Takalo M., Wittrahm R., Wefers B., Parhizkar S., Jokivarsi K., Kuulasmaa T., Mäkinen P., Martiskainen H., Wurst W., Xiang X., Marttinen M., Poutiainen P., Haapasalo A., Hiltunen M., Haass C., The Alzheimer’s disease-associated protective Plcγ2-P522R variant promotes immune functions. Mol. Neurodegener. 15, 1–14 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R24] 24.Maguire E., Menzies G. E., Phillips T., Sasner M., Williams H. M., Czubala M. A., Evans N., Cope E. L., Sims R., Howell G. R., Lloyd-Evans E., Williams J., Allen N. D., Taylor P. R., PIP2 depletion and altered endocytosis caused by expression of Alzheimer’s disease-protective variant PLCγ2 R522. EMBO J. 40, e105603 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R25] 25.Tsai A. P., Dong C., Lin P. B. C., Oblak A. L., Viana Di Prisco G., Wang N., Hajicek N., Carr A. J., Lendy E. K., Hahn O., Atkins M., Foltz A. G., Patel J., Xu G., Moutinho M., Sondek J., Zhang Q., Mesecar A. D., Liu Y., Atwood B. K., Wyss-Coray T., Nho K., Bissel S. J., Lamb B. T., Landreth G. E., Genetic variants of phospholipase C-γ2 alter the phenotype and function of microglia and confer differential risk for Alzheimer’s disease. Immunity 56, 2121–2136.e6 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R26] 26.Oblak A. L., Kotredes K. P., Pandey R. S., Reagan A. M., Ingraham C., Perkins B., Lloyd C., Baker D., Lin P. B., Soni D. M., Tsai A. P., Persohn S. A., Bedwell A. A., Eldridge K., Speedy R., Meyer J. A., Peters J. S., Figueiredo L. L., Sasner M., Territo P. R., Sukoff Rizzo S. J., Carter G. W., Lamb B. T., Howell G. R., Plcg2M28L interacts with high fat/high sugar diet to accelerate alzheimer’s disease-relevant phenotypes in mice. Front. Aging Neurosci. 14, 886575 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R27] 27.Hajicek N., Keith N. C., Siraliev-Perez E., Temple B. R. S., Huang W., Zhang Q., Harden T. K., Sondek J., Structural basis for the activation of plc-γ isozymes by phosphorylation and cancer-associated mutations. Elife 8, e51700 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R28] 28.Kim H. K., Kim J. W., Zilberstein A., Margolis B., Kim J. G., Schlessinger J., Rhee S. G., PDGF stimulation of inositol phospholipid hydrolysis requires PLC-γ1 phosphorylation on tyrosine residues 783 and 1254. Cell 65, 435–441 (1991). [DOI] [PubMed] [Google Scholar]

[R29] 29.Gresset A., Hicks S. N., Harden T. K., Sondek J., Mechanism of phosphorylation-induced activation of phospholipase C-γ isozymes. J. Biol. Chem. 285, 35836–35847 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R30] 30.Liu Y., Bunney T. D., Khosa S., Macé K., Beckenbauer K., Askwith T., Maslen S., Stubbs C., de Oliveira T. M., Sader K., Skehel M., Gavin A. C., Phillips C., Katan M., Structural insights and activating mutations in diverse pathologies define mechanisms of deregulation for phospholipase C gamma enzymes. EBioMedicine 51, 102607 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R31] 31.Bae J. H., Lew E. D., Yuzawa S., Tomé F., Lax I., Schlessinger J., The selectivity of receptor tyrosine kinase signaling is controlled by a secondary SH2 domain binding site. Cell 138, 514–524 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R32] 32.Huang Z., Marsiglia W. M., Basu Roy U., Rahimi N., Ilghari D., Wang H., Chen H., Gai W., Blais S., Neubert T. A., Mansukhani A., Traaseth N. J., Li X., Mohammadi M., Two FGF receptor kinase molecules act in concert to recruit and transphosphorylate phospholipase Cγ. Mol. Cell 61, 98–110 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R33] 33.Peters K. G., Marie J., Wilson E., Ives H. E., Escobedo J., Del Rosario M., Mirda D., Williams L. T., Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca²⁺ flux but not mitogenesis. Nature 358, 678–681 (1992). [DOI] [PubMed] [Google Scholar]

[R34] 34.Mohammadi M., Dionne C. A., Li W., Li N., Spivak T., Honegger A. M., Jaye M., Schlessinger J., Point mutat ion in FGF receptor eliminates phosphatidylinositol hydrolysis without affecting mitogenesis. Nature 358, 681–684 (1992). [DOI] [PubMed] [Google Scholar]

[R35] 35.Watanabe D., Hashimoto S., Ishiai M., Matsushita M., Baba Y., Kishimoto T., Kurosaki T., Tsukada S., Four tyrosine residues in phospholipase C-γ2, identified as Btk-dependent phosphorylation sites, are required for B cell antigen receptor-coupled calcium signaling. J. Biol. Chem. 276, 38595–38601 (2001). [DOI] [PubMed] [Google Scholar]

[R36] 36.Kim Y. J., Sekiya F., Poulin B., Bae Y. S., Rhee S. G., Mechanism of B-cell receptor-induced phosphorylation and activation of phospholipase C-γ2. Mol. Cell. Biol. 24, 9986–9999 (2004). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R37] 37.Özdener F., Dangelmaier C., Ashby B., Kunapuli S. P., Daniel J. L., Activation of phospholipase Cγ2 by tyrosine phosphorylation. Mol. Pharmacol. 62, 672–679 (2002). [DOI] [PubMed] [Google Scholar]

[R38] 38.Huang W., Carr A. J., Hajicek N., Sokolovski M., Siraliev-Perez E., Hardy P. B., Pearce K. H., Sondek J., Zhang Q., A high-throughput assay to identify allosteric inhibitors of the PLC-γ isozymes operating at membranes. Biochemistry 59, 4029–4038 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R39] 39.Jumper J., Evans R., Pritzel A., Green T., Figurnov M., Ronneberger O., Tunyasuvunakool K., Bates R., Žídek A., Potapenko A., Bridgland A., Meyer C., Kohl S. A. A., Ballard A. J., Cowie A., Romera-Paredes B., Nikolov S., Jain R., Adler J., Back T., Petersen S., Reiman D., Clancy E., Zielinski M., Steinegger M., Pacholska M., Berghammer T., Bodenstein S., Silver D., Vinyals O., Senior A. W., Kavukcuoglu K., Kohli P., Hassabis D., Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R40] 40.Varadi M., Anyango S., Deshpande M., Nair S., Natassia C., Yordanova G., Yuan D., Stroe O., Wood G., Laydon A., Zídek A., Green T., Tunyasuvunakool K., Petersen S., Jumper J., Clancy E., Green R., Vora A., Lutfi M., Figurnov M., Cowie A., Hobbs N., Kohli P., Kleywegt G., Birney E., Hassabis D., Velankar S., AlphaFold protein structure database: Massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 50, D439–D444 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R41] 41.Aslanidis C., de Jong P. J., Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18, 6069–6074 (1990). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R42] 42.Siraliev-Perez E., Stariha J. T. B., Hoffmann R. M., Temple B. R. S., Zhang Q., Hajicek N., Jenkins M. L., Burke J. E., Sondek J., Dynamics of allosteric regulation of the phospholipase C-γ isozymes upon recruitment to membranes. Elife 11, e77809 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R43] 43.Gasteiger E., Gattiker A., Hoogland C., Ivanyi I., Appel R. D., Bairoch A., ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 31, 3784–3788 (2003). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R44] 44.Wang Q. S., Yu F., Huang S., Sun B., Zhang K. H., Liu K., Wang Z. J., Xu C. Y., Wang S. S., Yang L. F., Pan Q. Y., Li L., Zhou H., Cui Y., Xu Q., Earnest T., He J. H., The macromolecular crystallography beamline of SSRF. Nucl. Sci. Tech. 26, 1–6 (2015). [Google Scholar]

[R45] 45.Vonrhein C., Flensburg C., Keller P., Sharff A., Smart O., Paciorek W., Womack T., Bricogne G., Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. Sect. D Biol. Crystallogr. 67, 293–302 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R46] 46.Kabsch W., XDS. Acta Crystallogr D Biol Crystallogr 66, 125–132 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R47] 47.Winn M. D., Ballard C. C., Cowtan K. D., Dodson E. J., Emsley P., Evans P. R., Keegan R. M., Krissinel E. B., Leslie A. G. W., McCoy A., McNicholas S. J., Murshudov G. N., Pannu N. S., Potterton E. A., Powell H. R., Read R. J., Vagin A., Wilson K. S., Overview of the CCP4 suite and current developments. Acta Crystallogr. Sect. D Biol. Crystallogr. 67, 235–242 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R48] 48.McCoy A. J., Grosse-Kunstleve R. W., Adams P. D., Winn M. D., Storoni L. C., Read R. J., Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R49] 49.Murshudov G. N., Skubák P., Lebedev A. A., Pannu N. S., Steiner R. A., Nicholls R. A., Winn M. D., Long F., Vagin A. A., REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. Sect. D Biol. Crystallogr. 67, 355–367 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R50] 50.Emsley P., Lohkamp B., Scott W. G., Cowtan K., Features and development of Coot. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 486–501 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R51] 51.The PyMOL Molecular Graphics System, Version 2.3 Schrödinger LLC.

[R52] 52.Zheng S. Q., Palovcak E., Armache J. P., Verba K. A., Cheng Y., Agard D. A., MotionCor2: Anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R53] 53.Rohou A., Grigorieff N., CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R54] 54.Ru H., Chambers M. G., Fu T. M., Tong A. B., Liao M., Wu H., Molecular mechanism of V(D)J recombination from synaptic RAG1-RAG2 complex structures. Cell 163, 1138–1152 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R55] 55.Zivanov J., Nakane T., Forsberg B. O., Kimanius D., Hagen W. J. H., Lindahl E., Scheres S. H. W., New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife 7, e42166 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R56] 56.Punjani A., Rubinstein J. L., Fleet D. J., Brubaker M. A., CryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017). [DOI] [PubMed] [Google Scholar]

[R57] 57.Afonine P. V., Poon B. K., Read R. J., Sobolev O. V., Terwilliger T. C., Urzhumtsev A., Adams P. D., Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. Sect. D Struct. Biol. 74, 531–544 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R58] 58.Pettersen E. F., Goddard T. D., Huang C. C., Meng E. C., Couch G. S., Croll T. I., Morris J. H., Ferrin T. E., ChimeraX U. C. S. F., Structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R59] 59.Emsley P., Cowtan K., Coot: Model-building tools for molecular graphics. Acta Crystallogr. Sect. D Biol. Crystallogr. 60, 2126–2132 (2004). [DOI] [PubMed] [Google Scholar]

[R60] 60.Carr A. J., Siraliev-Perez E., Huang W., Sondek J., Zhang Q., Fluorogenic XY-69 in lipid vesicles for measuring activity of phospholipase C isozymes. Methods Mol. Biol. 2251, 225–236 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R61] 61.Jing H., Reed A., Ulanovskaya O. A., Grigoleit J. S., Herbst D. M., Henry C. L., Li H., Barbas S., Germain J., Masuda K., Cravatt B. F., Phospholipase Cγ2 regulates endocannabinoid and eicosanoid networks in innate immune cells. Proc. Natl. Acad. Sci. U.S.A. 118, e2112971118 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

The crystal and cryo-EM structures of PLCγ2 reveal dynamic interdomain recognitions in autoinhibition

Young-Cheul Shin

Ashlee Marie Plummer-Medeiros

Alison Mungenast

Hyeong-wook Choi

Karen TenDyke

Xiaojie Zhu

Jennifer Shepard

Kristen Sanders

Ningning Zhuang

Liang Hu

Dongming Qian

Kangkang Song

Chen Xu

John Wang

Suresh B Poda

Maofu Liao

Yu Chen

Roles

Abstract

INTRODUCTION

RESULTS

Crystal structure of human PLCγ2

Fig. 1. Crystal structure of human PLCγ2.

Disease-associated mutations and variants of PLCγ2

Fig. 2. Positions and mapping of PLCγ2 mutations and variants.

Structure analysis by single-particle cryo-EM

Cryo-EM structures of PLCγ2

Fig. 3. Cryo-EM structure of full-length human PLCγ2 (PLCγ2-F).

Fig. 4. Cryo-EM structure of full-length PLCγ2 containing visible C-terminal helical structure, PLCγ2-H.

Cryo-EM structure of PLCγ2 in complex with FGFR1K

Fig. 5. Cryo-EM structure of human PLCγ2 in complex with pFGFR1K.

DISCUSSION

Fig. 6. Dynamic interactions in autoinhibited states of PLCγ2 and recognition by receptor kinase FGFR1.

MATERIALS AND METHODS

Experimental design

Protein cloning, expression, and purification

Crystallization

Diffraction data collection and crystal structure determination

Cryo-EM sample preparation and data collection

Cryo-EM data processing

Cryo-EM structure model building, refinement, and display

Size exclusion chromatography examining PLCγ2/FGFR1K association

Lipase activity measurement

Acknowledgments

Supplementary Materials

This PDF file includes:

REFERENCES AND NOTES

Associated Data

Supplementary Materials

ACTIONS

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