Fidelity of G protein β-subunit association by the G protein γ-subunit-like domains of RGS6, RGS7, and RGS11

Abstract

Several regulators of G protein signaling (RGS) proteins contain a G protein γ-subunit-like (GGL) domain, which, as we have shown, binds to G_β5 subunits. Here, we extend our original findings by describing another GGL-domain-containing RGS, human RGS6. When RGS6 is coexpressed with different G_β subunits, only RGS6 and G_β5 interact. The expression of mRNA for RGS6 and G_β5 in human tissues overlaps. Predictions of α-helical and coiled-coil character within GGL domains, coupled with measurements of G_β binding by GGL domain mutants, support the contention that G_γ-like regions within RGS proteins interact with G_β5 subunits in a fashion comparable to conventional G_β/G_γ pairings. Mutation of the highly conserved Phe-61 residue of G_γ2 to tryptophan, the residue present in all GGL domains, increases the stability of the G_β5/G_γ2 heterodimer, highlighting the importance of this residue to GGL/G_β5 association.

The “regulators of G protein signaling” (RGS) protein family consists of at least 20 mammalian gene products that act as GTPase activating proteins on the α-subunits of heterotrimeric, signal-transducing G proteins (1–3). By accelerating the inactivation of GTP-bound G_α subunits, RGS proteins serve as negative regulators of G protein-mediated signaling pathways. Additionally, two RGS proteins, p115–RhoGEF and PDZ–RhoGEF, can also act as effectors, coupling GTP-bound G_α12 and/or G_α13 to Rho activation (4, 5).

Regions within certain RGS proteins that lie outside the RGS domain interact with other components of G protein signal transduction machinery. For example, the N-terminal PDZ domain of RGS12 associates in vitro with the C termini of G protein-coupled receptors (6), and the N-terminal domain of RGS4 mediates receptor-selective inhibition of G protein-mediated Ca²⁺ signaling in pancreatic acinar cells (7). We recently identified a G protein γ subunit-like (GGL) domain within four mammalian RGS family members and identified its role in mediating specific interaction of RGS7 and RGS11 with G protein β5-subunits (8). In a complementary study, a native RGS7/G_β5 complex has been isolated from bovine retina (9). Our discovery of RGS7/G_β5 and RGS11/G_β5 associations tripled the number of known interacting partners for this outlier G_β subunit, which previously had been known to interact only with G_γ2 (10–13). Here, we describe the cloning, expression, and G_β binding selectivity of RGS6, another RGS protein possessing the GGL domain. Residues critical for G_β subunit binding specificity have been identified by mutagenesis of G_γ2 and the GGL domains of RGS6, RGS7, and RGS11.

MATERIALS AND METHODS

Expression Constructs.

cDNAs for RGS7, RGS11, and G protein subunits have been described (8). RGS6 was isolated by reverse transcription–PCR by using sense (5′-GCG GCC GCA TGG CTC AAG GAT CCG GGG ATC AAA G-3′) and antisense (5′-TCT AGA CTG GGA TCA GGG CCT CTT AGC GAG-3′) primers as described (14) and subcloned with an N-terminal hemagglutinin (HA)-epitope tag into pcDNA3.1 (Invitrogen). G protein β-subunit cDNAs were subcloned with an N-terminal myc-epitope tag into pcDNA3.1 (Invitrogen). Mutagenesis was performed as described (6). The G_γ2 (F61W)/RGS fusion was created by adding a 17-aa linker (PRAAASVMDICRIRPWYP; derived from pcDNA3.1 polylinker) C-terminal to amino acid 70 of the G_γ2 (F61W) point mutant, followed by the RGS domain of rat RGS12 (amino acids 716–838; GenBank acc. no. U92280).

In Vitro Transcription/Translation.

Reactions were performed, and translation products were immunoprecipitated and analyzed by SDS/PAGE as described (8), except for variations in detergent conditions. “Low-detergent” immunoprecipitations were performed and washed in buffer D [50 mM NaCl/10 mM MgCl₂/50 mM Tris, pH 8.0/1 mM EDTA/10 mM 2-mercaptoethanol/20% (vol/vol) glycerol/Complete protease inhibitors; Roche Diagnostics] containing 0.05% C12E10, whereas “high-detergent” immunoprecipitations were performed in buffer D containing 0.1% Triton X-100 and washed in RIPA-500 buffer containing 500 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, and 0.1% SDS.

Transient Transfection and Immunoprecipitation.

COS-7 cell lysates were prepared and immunoprecipitated by using RIPA-150 buffer as described (8); proteins were separated by SDS/PAGE, electroblotted onto nitrocellulose, and detected with primary antibodies conjugated to horseradish peroxidase (HRP) and chemiluminescence (Amersham Pharmacia). The HRP-conjugated anti-HA mAb 3F10 was obtained from Roche Diagnostics; HRP-conjugated anti-myc mAb was purchased from Invitrogen.

Model Generation and Analysis.

Published alignments (15) allowed for replacement of residues at the interface of G_β1 and G_γ1 with the corresponding residues of G_β5 and the GGL domain of hRGS11 by using the program o (16). Where necessary, alternate allowed rotamers were chosen to limit steric overlap. Conjugate gradient energy minimization of the initial model was done with x-plor (17) by using standard energy functions and a coordinate restraint term applied to Cα-positions to compensate for the lack of experimental restraints. Cavity determination was performed by using the program voidoo (18). Structures were presented by using insight (Molecular Simulations, Waltham, MA). Secondary structure and coiled-coil predictions were performed with predictprotein (www.embl-heidelberg.de; ref. 19).

RESULTS AND DISCUSSION

RGS6 Has a DEP/GGL/RGS Domain Structure and Binds G_β5.

To extend our findings on the G_β binding specificity of RGS7 and RGS11 to other uncharacterized RGS proteins, we cloned human RGS6. A partial clone of RGS6 was described originally as S194, a brain-derived mRNA identified during mapping of the human AD3 locus at chromosome 14q24.3 (20). Database searches with S194 identified additional human RGS6 sequence records (e.g., GenBank acc. nos. H09621, AA351873, and AF073920). Oligonucleotides flanking the predicted ORF of full-length RGS6 were used to amplify RGS6 mRNA by reverse transcription–PCR from human brain total RNA. Two forms of RGS6 cDNA were isolated (GenBank acc. nos. AF107619 and AF107620), encoding ORFs of 490 or 472 aa, respectively; the difference between forms is the presence or absence of 18 contiguous amino acids (457-PESEQGRRTSLEKFTRSV-474) C-terminal to the RGS domain, likely a result of alternative splicing.

RGS6 is most similar to RGS7 (21–23). Both RGS6 isoforms encode an N-terminal DEP (Disheveled, EGL-10, Pleckstrin) domain (amino acids 39–121) 81% identical to the mouse RGS7 DEP domain, a C-terminal RGS domain (amino acids 333–447) 80% identical to the RGS box of human RGS7, and a GGL domain (amino acids 254–317) 57% identical to the mouse RGS7 GGL domain. In comparison to G_γ subunits, the central portion of the RGS6 GGL domain (amino acids 262–309) is most similar to bovine G_γ2 (41% identity).

As the RGS7 and RGS11 GGL domains bind only G_β5 subunits (8), we tested the RGS6 GGL domain for G_β binding specificity. G_β subunits (G_β1–G_β5) were produced by in vitro transcription/translation along with HA-epitope-tagged RGS proteins or G_γ subunits to detect possible interacting pairs. An anti-HA mAb was used to immunoprecipitate ³⁵S-labeled G_γ and RGS proteins; associated ³⁵S-labeled G_β subunits were detected by SDS/PAGE and autoradiography. G_γ2 bound to G_β1, G_β2, and G_β4 but not G_β5 (Fig. 1A), as previously described (8, 24). The G_β5/G_γ2 complex is reported to be abnormally sensitive to low levels of detergent compared with other G_β/G_γ pairings (25); we therefore believe that the minimal detergent (0.05% C12E10) necessary during immunoprecipitation results in disruption of the G_β5/G_γ2 complex.

Figure 1.

G_β binding specificity of RGS6 and RGS7. G_β subunits were cotranslated in reticulocyte lysates with (A) HA-tagged G_γ2 or (B) HA-tagged, truncated RGS6 (ΔDΔC, where ΔD indicates a DEP domain deletion and ΔC indicates a C-terminal deletion; amino acids 255–456) or truncated RGS7 (ΔDΔC; amino acids 202–395, SwissProt P49802) protein. Lysates were immunoprecipitated (IP) in low detergent with anti-HA mAb, and immunoprecipitated proteins and clarified supernatants were visualized separately by SDS/PAGE and autoradiography. (C) Expression vectors for full-length, HA-tagged RGS6 and individual, myc-tagged G_β subunits were transiently cotransfected into COS-7 cells. Cell lysates were immunoprecipitated with anti-HA mAb, and coimmunoprecipitated G_β subunits were detected by immunoblotting (Blot) with anti-myc-HRP or anti-HA-HRP conjugates.

In contrast to the G_β binding specificity of G_γ2, a truncated RGS6 protein containing both GGL and RGS domains (RGS6ΔDΔC) did not interact with G_β1–G_β4 but bound G_β5, the same specificity observed with the analogously truncated RGS7 (Fig. 1B) and RGS11 (see Fig. 3D and ref. 8). To determine the specific nature of RGS6/G_β5 association in a cellular context, COS-7 cells were cotransfected with HA-tagged, full-length RGS6 and one of five myc-tagged G_β subunits. Lysates from transfected cells were immunoprecipitated with anti-HA mAb, and associated G_β subunits were detected with anti-myc antibody. Of the five G_β subunits tested, only G_β5 coimmunoprecipitated with full-length RGS6 (Fig. 1C). We routinely observe greater levels of RGS6 and G_β5 proteins in cell lysates when they are coexpressed, perhaps reflective of the instability of unpaired G_β and GGL subunits similar to the instability seen in classical G_β and G_γ subunits (26–28), including G_β5 (11).

Figure 3.

In vitro G_β binding specificity of GGL domain mutants. (A) Secondary structure predictions for the RGS6 GGL domain and sequence alignment between G_γ2, RGS6, RGS7, RGS9, and RGS11. Identical residues are in black boxes; conserved residues are shown in shaded boxes. For RGS6, probabilities of α-helical character (indexed to a maximum of 9; ref. 19) and coiled-coil interaction (indexed to a maximum of 1.0; ref. 33) are plotted above the primary sequence of the GGL domain (x axis). α-Helices within G_γ2 (ref. 32) are indicated by an α above the sequence. The position and nature of point mutations are denoted above or below the sequence line with arrows. Individual G_β subunits were cotranslated in reticulocyte lysates with wild-type or mutant RGS6 (B), RGS7 (C), and RGS11 proteins (D–F). HA-tagged RGS or G_γ proteins were immunoprecipitated in low detergent (except as noted in E) with anti-HA mAb, and associated G_β proteins were visualized by SDS/PAGE and autoradiography. (E) Immunoprecipitations (IP) of cotranslated G_β5 and wild-type (lane 1) or W274F mutant (lanes 2 and 3) RGS11ΔDΔC proteins were performed in high-detergent (lanes 1 and 3) or low-detergent (lane 2) conditions and visualized separately from clarified supernatants (Sup’nt) as above.

We conclude that, within the DEP/GGL/RGS subfamily of RGS proteins, RGS6 is most closely related to RGS7 in primary sequence and shares G_β5 binding selectivity with both RGS7 and RGS11.

RGS6 and G_β5 mRNAs Have Overlapping Distribution.

Unlike the widespread expression of other G_β subunits, G_β5 mRNA is expressed in a tissue-restricted manner: mouse G_β5 is expressed predominantly in the brain and retina (10, 29), whereas human G_β5 mRNA is found in brain, retina, kidney, and pancreas (8, 30). To test whether RGS6 and G_β5 are coexpressed in human tissues, we compared expression patterns of both genes in Northern blot analyses. RGS6 mRNA was detected in whole brain (Fig. 2 A and B) and in brain anatomical regions with an expression pattern overlapping that of G_β5; however, RGS6 expression is barely detectable in the caudate putamen and spinal cord (Fig. 2C). This pattern of expression is in contrast with that reported by Gold et al. (31) for RGS6 in the rat brain; such a cross-species difference in brain expression patterns also was observed for RGS11 (8, 31). Unlike the expression pattern of human RGS11, human RGS6 mRNA is not seen in the retina or pancreas but is observed in the heart (Fig. 2 A and B). The observed coexpression of RGS6 and G_β5 mRNA in the human brain suggests that an RGS6/G_β5 complex may play a role in G protein-mediated neuronal signaling.

Figure 2.

Comparison of RGS6 and G_β5 expression patterns. Northern blots of 20 μg total RNA (A) or 2 μg poly(A+) RNA from various human tissues (B and C) were serially hybridized with a G_β5 cDNA probe (ref. 8), with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe as a control for RNA loading and quality, and with an RGS6 cDNA probe. kb, kilobase.

GGL Domain Fidelity: Testing the “Trp-274 Hypothesis.”

An assumption inherent in our original report (8) is that sequence homology between GGL domains and G_γ subunits reflects a similarity at the secondary and quaternary structure levels—i.e., a predominantly α-helical extended chain forming extensive contacts, including an N-terminal parallel coiled coil (15), with G_β5 subunits. Indeed, secondary structure predictions for RGS6 (Fig. 3A), RGS7, and RGS11 (data not shown) suggest an α-helical character in regions analogous to the first two α-helices of G_γ1 and G_γ2 (15, 32); for the RGS9 GGL domain, only the N-terminal region (amino acids 216–233) is predicted to be α-helical (data not shown). Furthermore, for both RGS6 and RGS7, the putative N-terminal α-helix within the GGL domain is predicted to participate in a coiled-coil structure (Fig. 3A and data not shown); its putative counterpart, the N terminus of G_β5, is also predicted to form a coiled coil by the same program (ref. 33; data not shown).

In our initial report (8), we predicted an important role for Trp-274 of the RGS11 GGL domain in G_β5 selectivity (the Trp-274 hypothesis), given that (i) all G_γ subunits have a smaller aromatic residue (Phe) at the analogous position and (ii) G_β1–G_β4 have a larger residue (Asn-340) than that of G_β5 (Ala-353) at the position predicted to interact with Trp-274 of RGS11 (8). To test this prediction, we created GGL-domain mutants by replacing tryptophan with phenylalanine in RGS6 (W309F), RGS7 (W306F), and RGS11 (W274F) and assessed G_β binding by coexpression with G_β1–G_β5 in vitro. None of the Trp-to-Phe mutants acquired affinity toward G_β1–G_β4 (Fig. 3 B–D), and the mutations had only a modest impact on G_β5 affinities in low-detergent conditions (0.05% C12E10). However, the Trp-to-Phe mutations destabilized the GGL/G_β5 interactions in high-detergent conditions; for example, when washed with 1% Triton X-100/0.1% SDS/0.5% sodium cholate, very little G_β5 remained bound to the W274F mutant of RGS11 (Fig. 3E, lane 3) in comparison to robust G_β5 binding observed for the wild-type protein (Fig. 3E, lane 1). Similarly destabilized G_β5 interactions were observed with RGS6 and RGS7 Trp-to-Phe mutants in high-detergent conditions (data not shown). This region is clearly critical for G_β binding, as mutation of tryptophan and the preceding proline to serine and alanine, respectively, eliminates G_β5 binding by RGS11 (PW274AS; Fig. 3D). This result is entirely consistent with the known importance of this dipeptide motif in the G_β1/G_γ1 and G_β1/G_γ2 crystal structures, in which the proline “kinks” the Cα-chain inward toward G_β1, positioning the phenylalanine residue into a hydrophobic pocket (15, 32).

GGL Domain Fidelity: Role of Other Residues.

Other amino acids within RGS6 and RGS11 were also modified, singly or in tandem, to test their roles in both G_β5 binding affinity and G_β subunit binding selectivity. Ser-245 of RGS11 and Asp-297 of RGS6 represent two positions conserved among all GGL domains and G_γ subunits (8). The analogous positions within G_γ1 (Ser-34 and Asp-51) and G_γ2 (Ser-31 and Asp-48) form hydrogen bonds with Asp-27 and Ser-279/Ser-281 of G_β1, respectively (15, 32). Conversion of Ser-245 (RGS11) and Asp-297 (RGS6) to alanine confirmed their importance to GGL domain function; considerably reduced G_β5 binding was seen with the RGS11 S245A mutant (Fig. 3F), whereas the RGS6 D297A mutant did not bind G_β5 at all, either in vitro (Fig. 3B) or in cell cotransfection studies (data not shown). In contrast, Gln-271 of RGS6 is conserved only in the highly related RGS7; as expected, mutation of Gln-271 to alanine (the residue present within RGS9 and RGS11) had no discernable effect on G_β5 binding by RGS6 (Fig. 3B).

Gln-257 of RGS11 is conserved among all GGL domains, including that of EGL-10 (8), whereas the analogous position within G_γ1 (Glu-46) and G_γ2 (Ala-43) is neither well conserved nor involved in interactions with G_β1 (15, 32). Hence, as predicted by the lack of importance to G_γ/G_β interactions, replacement of Gln-257 with alanine yielded no change in the G_β binding behavior of RGS11 (Q257A; Fig. 3F). To this point, therefore, GGL domain mutations affect GGL/G_β5 assembly when made in positions analogous to G_γ/G_β contact points. Conversely, GGL domain mutations do not affect GGL/G_β5 assembly when made in positions analogous to those not involved in G_γ/G_β contacts. These findings support our assumption that the GGL domain interacts with G_β5 in a manner similar to conventional G_γ/G_β pairings.

GGL Domain Fidelity: Comparison to G_γ1’s Selectivity for G_β1.

Studies of G_γ1 and G_γ2 binding specificities have identified a five-residue region of G_γ1 (amino acids 36–40) critical for selective dimerization with G_β1 and not G_β2 (34, 35); replacement of three residues of G_γ1, Cys-36–Cys-37–Glu-38, with the corresponding residues of G_γ2, Ala-33–Ala-34–Ala-35, is sufficient to allow binding of G_γ1 to G_β2 (35). Therefore, we replaced the corresponding residues of RGS11 (Cys-247–Leu-248–Glu-249) with three alanines and tested G_β subunit association. The triple-alanine mutation, either alone (CLE249AAA) or in combination with mutation to Trp-274 (W274F + CLE249AAA; not shown), neither engendered additional G_β binding specificity nor abrogated association with G_β5 (Fig. 3F) and G_β5L (data not shown), suggesting that, unlike G_γ1 and G_γ2, this region of the GGL domain does not control G_β binding selectivity.

Fidelity of G_γ1 and G_γ2: Revisiting the Trp-274 Hypothesis.

We tested a corollary of the Trp-274 hypothesis espoused above; namely, that conversion of the analogous position within G_γ1 and G_γ2 from phenylalanine to tryptophan would prevent or weaken binding to G_β1–G_β4 and/or allow G_β5 association. The G_β binding characteristics of G_γ1 were unaffected by this mutation (F64W), as compared with wild-type G_γ1 (Fig. 4A). In contrast, the same mutation to G_γ2 (F61W), although it did not affect affinity toward G_β1–G_β4, engendered binding to G_β5 when tested in vitro in low-detergent conditions (Fig. 4A). Under high-detergent conditions, G_β5 binding was not detected, whereas binding to other G_β subunits was unaffected [G_γ2 (F61W) RIPA-500 vs. C12E10; Fig. 4A].

Figure 4.

G_β binding specificity of G_γ1 and G_γ2 mutants. HA-tagged G_γ proteins (wild-type or mutated as indicated) were either cotranslated in vitro (A and C) or cotransfected into COS-7 cells (B) with individual G_β subunits, immunoprecipitated (IP) with anti-HA mAb in either low detergent (0.05% C12E10) or high detergent (RIPA), and visualized by SDS/PAGE and autoradiography (A and C) or immunoblotting (B) with indicated antisera (Blot). “Fusion” denotes chimeric protein composed of HA-tagged G_γ2 (F61W) subunit fused to the rat RGS12 RGS domain.

Of all mutants tested for G_β5 interaction, only the G_γ2 (F61W) mutant and the cognate GGL domain Trp-to-Phe mutants were affected by detergent conditions; all other GGL domain mutants (Fig. 3A) had the same affinity (or lack thereof) for G_β5 isoforms in cotranslation studies with low- or high-detergent conditions (data not shown). We believe this detergent sensitivity explains our inability to detect G_β5 binding by G_γ2 (F61W) after exposure to RIPA-150 buffer in cell cotransfection experiments (Fig. 4B). This conclusion is consistent with the extreme detergent sensitivity of the wild-type G_β5/G_γ2 complex (25). Additionally, in vitro cotranslation/immunoprecipitation studies with high-detergent RIPA-500 buffer disrupt RGS7/G_β5 association in contrast to robust interaction observed in low-detergent conditions (Fig. 1B and data not shown). The in vitro association of RGS6 and RGS11 with G_β5 isoforms has far more tolerance to high-detergent conditions, allowing for detection of such complexes upon COS-7 cotransfection and RIPA-buffer lysis (Fig. 1C and ref. 8).

Lack of Steric Hindrance from the RGS Domain.

To test the possibility that GGL-domain-containing RGS proteins are prevented from binding G_β1–G_β4 because of steric hindrance from a C-terminal RGS domain absent in conventional G_γ subunits, we created a chimeric G_γ2 (F61W)/RGS protein that mimics the spatial orientation of GGL and RGS domains. As observed for the G_γ2 (F61W) protein, the larger G_γ2 (F61W)/RGS12 fusion protein associated with G_β1, G_β2, G_β4, and G_β5 subunits in vitro in low-detergent conditions, with a loss only of G_β5 association apparent in high-detergent conditions (Fig. 3C). This result suggests that the lack of interaction between G_β1–G_β4 subunits and GGL-domain-containing RGS proteins is not the result of steric hindrance from the RGS domain but rather is intrinsic to the GGL domain.

Molecular Modeling of the Interaction of RGS11 Trp-274 with G_β5.

To understand the role of the Trp-274 position in differential G_β binding affinities of GGL domains and G_γ subunits, we returned to our RGS11/G_β5 interface model (8), which is based on side-chain replacement of the G_β1/G_γ1 crystal coordinates (15). The highlighted G_β5/GGL interface (Fig. 5B) has greater complementarity than the equivalent region in G_β1/G_γ1 (Fig. 5A). In the G_β1/G_γ1 crystal structure (15), packing defects around Phe-64 produce a ≈3.0-ų cavity, whereas no equivalent cavity surrounds Trp-274 in the modeled G_β5/GGL complex. In addition, Thr-338 and Ala-353 in G_β5 are smaller than the corresponding residues of G_β1 (Met-325 and Asn-340) and serve to accommodate the larger bulk of Trp-274 relative to Phe-64. Further enhancing the specificity of the G_β5/GGL complex, the hydroxyl group of Ser-92 in G_β5 potentially forms a hydrogen bond with the indole nitrogen of Trp-274; no similar hydrogen bond is possible in the G_β1/G_γ1 structure.

Figure 5.

Specificity-determining residues at the interface of G_β1 and G_γ1 compared with equivalent regions of modeled G_β5 and the GGL domain of RGS11. Highlighted in blue are van der Waals surfaces of G_β1 contacting Phe-64 of G_γ1 (A) or similar contacts between G_β5 and Trp-274 of the RGS11 GGL domain (B). Residues colored red in the G_β1/G_γ1 structure differ from equivalent residues in the G_β5/GGL model. Except for the conserved tripeptide motif (NPF or NPW), thin red and green lines trace the Cα-backbone of G_β1/G_γ1 and G_β5/GGL, respectively. Just before the conserved tripeptide motif, the Cα-traces diverge because of the insertion of a single residue in G_γ1 relative to the GGL domain.

The presence of a small cavity in G_β1/G_γ1 around Phe-64 suggests that this pocket could accommodate a larger residue, thereby allowing G_γ1 (F64W) and G_γ2 (F61W) mutants to maintain binding to their normal G_β partners (Fig. 4A). If this pocket within G_β5 specifies a Trp residue in GGL domains by virtue of increased cavity size and potential hydrogen bonding with Ser-92, then this specification could partially explain why G_γ2 (F61W) has increased affinity for G_β5 as a binding partner. The reverse mutation to Phe in the GGL domain would not fit as snugly into the pocket specifying Trp, thereby giving a relative loss of binding energy, which was observed as lowered in vitro affinity for G_β5 by RGS6(W309F), RGS7(W306F), and RGS11(W274F) under high-detergent conditions. We await experimentally derived structural data on the GGL/G_β5 complex to confirm the relative importance of this region to G_β binding specificity.

We extend our discovery of a GGL domain within RGS7 and RGS11 to include RGS6. Structural predictions and mutagenesis data strengthen our belief that the GGL domain binds to G_β5 subunits in a manner similar to that of conventional G_β/G_γ associations. The molecular basis for the absolute selectively of the GGL domain for G_β5 subunits remains to be determined. However, we have been able to increase the relative strength of the G_β5/G_γ2 association by mutating a single residue within G_γ2, Phe-61. This mutation is presumed to mimic atomic interactions unique to wild-type G_β5/GGL domain associations. Continued study of the nature of G_β5 association by GGL domains relative to that of Gγ subunits should assist our understanding of the role(s) of the DEP/GGL/RGS proteins in modulating G protein-coupled signaling.

ABBREVIATIONS

ΔD

DEP domain deletion

ΔC

C-terminus deletion

GGL

G protein γ-subunit-like

HA

hemagglutinin

HRP

horseradish peroxidase

Note

While this manuscript was under review, two additional reports of RGS/G_β5 association were published (36, 37). One reported the discovery of a tight association between RGS9 and G_β5L in rod photoreceptors (36). This discovery validates our original prediction based on the presence of a GGL domain within the primary sequence of RGS9.