Heterotrimeric G Protein Signaling Outside the Realm of Seven Transmembrane Domain Receptors

Abstract

Heterotrimeric G proteins, consisting of the guanine nucleotide-binding Gα subunits with GTPase activity and the closely associated Gβ and Gγ subunits, are important signaling components for receptors with seven transmembrane domains (7TMRs). These receptors, also termed G protein-coupled receptors (GPCRs), act as guanine nucleotide exchange factors upon agonist stimulation. There is now accumulating evidence for noncanonical functions of heterotrimeric G proteins independent of 7TMR coupling. Gα proteins belonging to all 4 subfamilies, including G_s, G_i, G_q, and G₁₂ are found to play important roles in receptor tyrosine kinase signaling, regulation of oxidant production, development, and cell migration, through physical and functional interaction with proteins other than 7TMRs. Association of Gα with non-7TMR proteins also facilitates presentation of these G proteins to specific cellular microdomains. This Minireview aims to summarize our current understanding of the noncanonical roles of Gα proteins in cell signaling and to discuss unresolved issues including regulation of Gα activation by proteins other than the 7TMRs.

Heterotrimeric G proteins are characteristically activated by seven-transmembrane domain receptors (7TMRs), also known as G protein-coupled receptors (GPCRs). The binding of an agonist to the extracellular or transmembrane domains of a 7TMR induces conformational changes in the receptor, which then functions as a guanine nucleotide exchange factor (GEF) for the associated GDP-bound Gα subunit. The switch to the active GTP-bound state triggers Gα subunit release from the receptor, leading to dissociation of Gβγ from Gα proteins and activation or inhibition of downstream effectors (Janetopoulos et al., 2001; Cabrera-Vera et al., 2003). Increasing evidence also suggests that, in some cases, upon ligand binding, the G proteins remain in the heterotrimeric form but undergo conformational rearrangement (Bünemann et al., 2003; Digby et al., 2006). The signaling cycle is complete after hydrolysis of GTP to GDP via the GTPase activity intrinsic to the Gα subunits. It is noteworthy that 7TMRs can signal independently of heterotrimeric G proteins. Several mechanisms mediating 7TMR signaling involve β-arrestins (Luttrell and Lefkowitz, 2002; Violin and Lefkowitz, 2007), G protein-coupled receptor kinases (Penn et al., 2000), JAKs/STATs (Marrero et al., 1995), Src-family tyrosine kinases (Sun et al., 1997; Thomas and Brugge, 1997), and scaffold proteins such as postsynaptic density 95/disc-large/zona occludens (PDZ) domain-containing proteins (Hall et al., 1998; Vila-Coro et al., 1999). In recent years, heterotrimeric G proteins have also emerged as noncanonical mediators of 7TMR-independent signaling pathways (Patel, 2004). This Minireview aims to address the pivotal roles of Gα proteins in regulating diverse biological responses through their interaction with proteins other than the 7TMRs.

Regulation of Gα Activation by Accessory Proteins

In recent years, novel accessory proteins involved in the activation of G proteins, either by directly influencing GTP binding or through their association with Gα or Gβγ subunits, have been uncovered (Sato and Ishikawa, 2010). Takesono et al. (1999) identified the activators of G protein signaling (AGS) that stimulate G protein activity independently of 7TMRs. The AGS group now contains 10 characterized members initially isolated on the basis of yeast functional screen (for review, see Blumer et al., 2007). The different AGS proteins exhibit selectivity for G protein subunits and present diverse modes of action ranging from promoting GTP binding as a GEF (AGS1 for Gα_i), stabilizing the GDP-bound complex as a guanine nucleotide dissociation inhibitor (AGS3 for Gα_i/o), and interfering with subunit interaction independently of nucleotide exchange (AGS8 with Gβγ). Although the signals integrated by these accessory proteins has not been fully delineated, they are part of the intracellular changes processed through G protein activation. AGS proteins might contribute to positioning G proteins and different effectors within the cell microdomains akin to a molecular scaffold. The growing list of nonreceptor GEFs includes Gα-interacting vesicle-associated protein (Garcia-Marcos et al., 2009), resistance to inhibitors of cholinesterase 8A (Tall et al., 2003), cysteine string protein (Natochin et al., 2005), and the yeast protein Arr4 (Lee and Dohlman, 2009).

Gα_i2 in its inactive (GDP-bound) state is found to associate with the cytosolic factor p67^phox of the NADPH oxidase (Marty et al., 2006). A possible consequence of this association is to target p67^phox to a specific subcellular compartment, thereby affecting NADPH oxidase assembly. Likewise, it is possible that mammalian AGS3 and Leu-Gly-Asn repeat-enriched proteins (Marty et al., 2003) and their Drosophila melanogaster homolog Pins, which contain an N-terminal tetratricopeptide repeat (TPR) domain, such as the one in p67^phox, have their activities directly regulated through association with Gα proteins. TPR domains were previously known for their interaction with small GTPases such as Rac (Das et al., 1998). Its binding to Gα proteins suggests a potentially novel regulatory mechanism for Gα activation independent of the 7TMRs. It is unclear, however, whether proteins bearing the TPR domains have the function of a GEF for Gα activation. Gα interaction with TPR domains may also facilitate its presentation to specific intracellular microdomains. Caveolin, a major component of caveolae membranes, has been found to copurify with multiple Gα proteins, including Gα_s, Gα_o, and Gα_i2 (Li et al., 1995). In this case, caveolin functions as a scaffolding protein to recruit Gα proteins to caveolin-rich areas of the plasma membrane.

Transactivation and Direct Activation of Gα Proteins through Receptor Tyrosine Kinases

Noncanonical activation of Gα proteins by receptor tyrosine kinases (RTKs) may be defined as either a direct activation process involving an interaction of the Gα protein with a RTK or functional cross-talk in the form of transactivation through the RTK (Patel, 2004). Agonists for growth factor receptors, including epidermal growth factor (EGF), fibroblast growth factor, platelet-derived growth factor (PDGF), insulin, insulin-like growth factor (IGF), and neurotrophins bind to RTKs, which are single-span transmembrane receptors. These agonists stimulate intrinsic tyrosine kinase activity encompassed in the cytoplasmic domains of the RTKs, resulting in receptor auto-phosphorylation on tyrosine residues and generation of docking sites for Src homology 2 domain-containing proteins such as Shc, Grb2, and phospholipase C-γ. Some of these growth factor receptors activate G proteins through functional cross-talk termed transactivation (Daub et al., 1996). It is increasingly evident that 7TMRs and RTKs form an integrated signaling network, and transactivation of RTKs by ligand-stimulated 7TMRs is a general process allowing for pleiotropic signaling (Lowes et al., 2002; Shah and Catt, 2004; Natarajan and Berk, 2006; Delcourt et al., 2007). EGFR is an extensively studied example of transactivation through RTKs (Daub et al., 1996) in which the activation of 7TMRs promotes production of EGF via metalloproteinase-catalyzed shedding of a transmembrane precursor and, in some cases, induction or tyrosine phosphorylation in the cytoplasmic tail of the RTK (Prenzel et al., 1999) (Fig. 1A). RTKs may in turn transactivate 7TMR signaling through physical association with Gα proteins, phosphorylation of the 7TMRs, and up-regulation of 7TMR ligand synthesis. Compelling evidence for the involvement of Gα proteins in RTK-mediated activation of various 7TMR effectors was provided with the use of pertussis toxin (PTX), which ADP-ribosylates the Gα_i/o family and uncouples these G proteins from their receptors, and cholera toxin, which alters the GTPase activity of Gα_s/olf (Gilman, 1987). Several studies have shown that the RTK ligand-activated events are sensitive to PTX (Table 1).

Fig. 1.

Schematic representation of selected noncanonical functions of Gα proteins. A, the EPOR requires association with the soluble tyrosine kinase JAK2 to mediate signaling (dotted arrow). The direct or indirect (dotted circle) interaction between the Gα_i protein and EPOR allows for EPO-induced activation of Gα_i and downstream effectors of both GTP-bound Gα_i and Gβγ, including transactivation through EGFR. B, the Toll-like receptor TLR4 mediates LPS signaling in collaboration with the glycosylphosphatidylinositol-anchored CD14 protein. The myeloid differentiation factor 88 (MyD88) and IL-1 receptor associated kinase (IRAK) are also involved in some of the TLR4 signaling pathways. The signal is relayed downstream by adaptor proteins and results in MAPK, AP-1 and nuclear factor-κB (NF-κB) activation (dotted arrow). The interaction between CD14 and Gα_i/o proteins regulates LPS-induced ERK activation and AP-1 transcriptional activity. C, the cell surface glycoprotein E-cadherin is linked to the cytoskeleton via its interaction with β-catenin, α-catenin and α-actinin. This interaction mediates cell-cell adhesion (dotted arrow). Gα_12/13 interaction with E-cadherin produces an allosteric effect (shaded box and trapezoid) uncoupling β-catenin from E-cadherin. Abolition of the E-cadherin/β-catenin complex disrupts cell adhesion and allows β-catenin, a known transcriptional regulator, to translocate into the nucleus. Not shown in this figure are the direct interaction of EGFR with Gα_s and Gα_s-mediated cAMP elevation, which was depicted in Fig. 2 of the review by Patel (2004), and the proposed signaling mechanism by which Gα_i1 and Gα_i3 mediate EGF-induced Akt-mTORC1 activation, which was described in the article by Cao et al. (2009). The Gα₁₃ involvement in αIIbβ3 integrin signaling was detailed in Fig. 4G of the article by Gong et al. (2010).

TABLE 1.

A nonexhaustive list of G proteins coupling to RTKs and their functions

RTK	G Proteins	Functions	References
EGFR	Gαi1/i2	Inhibition of adenylyl cyclase	Stryjek-Kaminska et al., 1996
	Gαi/o	Activation of PLC	Liang and Garrison, 1991
	Gαi1/i3/(i2?)	Activation of Akt-mTORC1 pathway	Cao et al., 2009
	Gαs	Activation of adenylyl cyclase	Nair et al., 1990
FGFR	Gαs	Activation of adenylyl cyclase
	Gβγ	Inhibition of NADPH oxidase	Krieger-Brauer et al., 2000
Insulin receptor	Gαi/o
	Gαq/11
	Gαi2	Activation of NADPH oxidase	Krieger-Brauer et al., 1997
IGF-IR	Gαi
	Gβγ		Luttrell et al., 1995
IGF-IIR	Gαi/o	Ca2+ influx and DNA synthesis	Okamoto et al., 1990
CSF-1R		PKC and Na+ influx	Imamura et al., 1990; Corre and Hermouet, 1995

FGFR, fibroblast growth factor receptor; CSF-1R, colony stimulating factor 1 receptor.

In addition to transactivation through RTKs, some of these receptors directly interact with Gα proteins (Patel, 2004). Early studies have shown that EGFR interacts with Gα_s (Nair et al., 1990; Poppleton et al., 1996) through its juxtamembrane region (Sun et al., 1997). This interaction leads to phosphorylation of Gα_s and elevation of intracellular cAMP in cardiac myocytes (Nair et al., 1990). The activation of Gα_s through EGFR is accompanied by augmented adenylyl cyclase activation and increased heart rate and contractility (Nair et al., 1993). It is noteworthy that EGFR also couples to Gα_i/o, and its choice for Gα_s or Gα_i/o coupling seems to be cell type-dependent. For example, EGF was found to induce hydrolysis of phosphatidylinositol 4,5-bisphosphate in rat hepatocytes, and this activity was abolished by PTX treatment (Liang and Garrison, 1991). Functional coupling of the Gα protein to EGFR was thought to be dependent on protein kinase C-induced phosphorylation of the receptor. Another single transmembrane domain receptor, IGF-II/mannose-6-phosphate receptor, was found to mediate endogenous acetylcholine release in a PTX-sensitive and PKC-dependent manner (Hawkes et al., 2006). Although no direct binding of Gα_i/o proteins to the IGF-II/mannose-6-phosphate receptor was investigated in this study, Okamoto et al. (1990) found that a part of the cytoplasmic domain (residues 2410–2423 located to the C-terminal end of the receptor) is responsible for interaction with Gα_i2. Upon receptor stimulation, Gα_i proteins are activated with the hierarchy of Gα_i2 > Gα_i1 ≈ Gα_i3. The activation of Gα_i proteins by a simple structure of the IGF-II/man6R is reminiscent to G protein activation by the amphiphilic peptide mastoparan (Higashijima et al., 1990). Similarities also exist between Gα_s interaction with the EGF RTKs and its interaction with the β2-adrenergic receptor (Okamoto et al., 1991; Sun et al., 1997). Overall, however, the exact mechanism of RTK-mediated G protein activation is not fully resolved and could depend on the RTK and the pathways activated by the ligand. In other reported cases, agonist stimulation of the insulin receptor leads to phosphorylation of Gα_i/o (Krupinski et al., 1988) and Gα_q/11 (Imamura et al., 1999). The tyrosine phosphorylation triggered through agonist binding to these receptors may promote the exchange of GDP for GTP on the Gα proteins. A partial list of RTKs that signal through Gα proteins is shown in Table 1.

Cao et al. (2009) reported recently that Gα_i1 and Gα_i3 are required for EGF-mediated activation of the Akt-mammalian target of rapamycin complex 1 (mTORC1) pathway. In this case, the Gα_i1 and Gα_i3 proteins form a complex with Grb2-associated binding protein 1 (Gab1), and these proteins are required for EGF-induced Gab1 phosphorylation and its subsequent interaction with the regulatory (p85) subunit of phosphatidylinositol 3-kinase (PI3K). As a result, EGF-induced cell migration and proliferation were compromised in the absence of Gα_i1 and Gα_i3. These results place Gα_i1 and Gα_i3 downstream of EGFR and upstream of Gab1. Exactly how the Gα_i proteins interact with Gab1 remains unclear, because PTX does not affect EGF-induced activation of the PI3K-Akt-mTORC1 pathway, indicating that a structure other than the C-terminal region of the Gα_i protein is involved in the interaction with Gab1. The role of the Gα_i proteins in EGFR signaling has some resemblance to that of the yeast Gα protein, which binds to the PI3K Vsp34 for its activation (Slessareva et al., 2006). However, the yeast Gpa1 protein translocates to endosome for this activity, whereas the Gα_i1 and Gα_i3 proteins remain close to the activated EGFR. Whether Gα_i2 has a similar function is unknown, because its absence did not affect EGF-induced Akt-mTORC1 activation (Cao et al., 2009). Deletion of the Gα_i1 and Gα_i3 genes did not affect EGF-induced activation of phospholipase C-γ and phosphorylation of STAT5, indicating that the Gα_i1 and Gα_i3 have a highly specialized function in EGFR signaling.

Role of Gα Proteins in Transmembrane Signaling through Other Non-7TMRs

A variety of cytokine receptors, including the receptors for erythropoietin (EPO), granulocyte macrophage-colony stimulating factor (GM-CSF), and interleukin-3 require members of the cytosolic tyrosine kinase JAK family for signaling. Ligand binding brings receptor-associated JAKs into apposition and leads to autophosphorylation of the JAK molecules and consequently to kinase activity. The activated JAK then phosphorylates specific tyrosine residues on the cytoplasmic tail of the receptor, creating docking sites for Src homology 2 domain-containing proteins such as the STATs. STATs are sequentially phosphorylated by JAKs on tyrosine residues then dissociated from the receptor to form active dimer protein complexes that translocate to the nucleus, where they bind to specific DNA sequences in promoters to modulate gene transcription. Other signaling pathways that are initiated by JAK activation include the PI3K and MAPK kinase pathways (Schindler, 2002). PTX has been shown to inhibit IL-3, GM-CSF and macrophage-colony stimulating factor signaling in hematopoietic cells (He et al., 1988; Imamura and Kufe, 1988; McColl et al., 1989). In these studies, it was not clear whether PTX influences the Gα_i-dependent, non-7TMR signaling cascades in a direct or indirect way, although the experimental data indicate that the PTX-sensitive substrate is a member of the G_i subfamily of G proteins. It is noteworthy that GM-CSF stimulation of neutrophils affects the distribution of Gα_i2 at the plasma membrane (Durstin et al., 1993). The increase in cell surface-associated Gα_i2 makes them more available for 7TMR signaling and is one of the known mechanisms for GM-CSF priming of neutrophils. Whether there is a direct coupling between GM-CSFR and Gα_i2 remains to be investigated. In the case of the EPOR, Guillard et al. (2001) discovered a novel EPO-dependent MAPK activation pathway involving the Gβγ subunit of G_i proteins. EPO treatment inhibited ADP-ribosylation of Gα_i and increased its binding of GTP. The activation of Gα_i and Gβγ suggests that the EPOR may catalyze guanine nucleotide exchange or trigger a transactivation mechanism.

Some receptors serving immune recognition functions are found to couple to G proteins. In independent studies conducted by DeFranco and colleagues, it was reported that B cell antigen receptor-mediated phosphoinositide signaling involves Gα proteins (Gold et al., 1987; DeFranco and Gold, 1989; DeFranco et al., 1989). However, the identity of the Gα proteins remains unclear, because the observed phosphoinositide signaling was not sensitive to PTX (Gold et al., 1987). An exception is that LPS signaling in the same B cell line and in macrophages was found to be sensitive to PTX inhibition (Jakway and DeFranco, 1986). Evidence for physical coupling of these receptors with the Gα proteins came from a study showing that Gα_i/o proteins coimmunoprecipitate with the glycosylphosphatidylinositol-anchored glycoprotein CD14, which is a critical component for LPS signaling through TLR4 (Solomon et al., 1998). The involvement of the G_i subfamily in LPS signaling was suggested in another study based on the observations that Gα_i2 and Gα_i3 minigenes (which block the functions of these Gα proteins) could block MAPK phosphorylation and AP-1 activation induced by a constitutively active TLR4 (Fan et al., 2004) (Fig. 1B). In some studies, the involvement of G_i class proteins in cytokine receptor signaling was suggested based on the inhibition by PTX, such as in IL-1R1 signaling through the serine/threonine kinase IRAK (Zumbihl et al., 1995; Cao et al., 1996). In these cases, whether there is physical association between the receptors and the G proteins is not entirely clear. In addition to the Gα_i proteins, Gα_q/11 have been found in complex with β-arrestin-1 and Src, which couple tumor necrosis factor-α to PI3K activation and inflammatory gene expression (Kawamata et al., 2007). Together, these findings suggest that proteins from the G_i and sometimes the G_q families may be important players in signaling through TLR4 and selected cytokine receptors.

Non-7TMRs that couple to Gα proteins also include the C-type natriuretic peptide receptor, amyloid precursor protein, T-cell receptors, and integrin-associated protein (CD47). An excellent review (Patel, 2004) summarizes the interactions between these non-7TMRs and the Gα proteins.

Noncanonical Functions of Gα_12/13 Proteins

Nearly all 7TM receptors that activate the G₁₂ subfamily (Gα₁₂ and Gα₁₃) also functionally couple to other subfamilies of Gα proteins, raising the intriguing questions of whether Gα₁₂ and Gα₁₃ are independently activated upon agonist binding to these receptors and whether they have functions different from those downstream of 7TM receptors. Research conducted thus far has produced a wealth of information supporting that idea the G₁₂ family of G proteins have noncanonical signaling properties (Table 2). Using yeast two-hybrid screening, a study showed that Gα₁₃ interacts with radixin, a member of the ERM (ezrin, radixin, and moesin) family of cytoskeleton-binding proteins (Vaiskunaite et al., 2000). Radixin interaction with activated Gα₁₃ increases its binding to polymerized F-actin, suggesting a possible role for Gα₁₃ in regulating the actin cytoskeleton. The Gα₁₂ and Gα₁₃ proteins also bind to the cytoplasmic domain of cadherins, causing dissociation of the transcriptional activator β-catenin from cadherins (Meigs et al., 2001). The epithelial intercellular adhesion molecule E-cadherin is linked to the cytoskeleton via its interaction with β-catenin (Fig. 1C). The association between E-cadherin and β-catenin plays a role in preventing β-catenin nuclear translocation and thus transcriptional activity. Because there is no direct competition between β-catenin and Gα₁₂ for binding to the same region of E-cadherin (Kaplan et al., 2001), the Gα₁₂-dependent dissociation of β-catenin from E-cadherin may result from an allosteric effect. This finding substantiates the well characterized transforming ability of the G₁₂ subfamily, because nuclear translocation of β-catenin is associated with increased transcriptional activation. Gα₁₂ and Gα₁₃ also regulate zebrafish epiboly, through a mechanism involving association with the cytoplasmic terminus of E-cadherin, which leads to inhibition of the activity of E-cadherin and cell adhesion (Lin et al., 2009).

TABLE 2.

A partial list of non-7TMR proteins that physically and/or functionally couple to the G₁₂ subfamily

Proteins	G Proteins	Functions	References
Cadherins	Gα12/13	Release of β-catenin for transcriptional activation; regulation of epiboly	Kaplan et al., 2001; Meigs et al., 2001; Lin et al., 2009
Factor VIIa/TF (tissue factor)	Gα12/13	Jak2 activation, Jak2/STAT5-dependent production of Bcl(XL)	Versteeg et al., 2004
Hax-1	Gα13	Gα13 forms complex with Hax-1, Rac and cortacin for cell movement	Radhika et al., 2004
Integrin aIIbβ3	Gα13	Integrin signaling, platelet adhesion	Gong et al., 2010
Hsp90	Gα12 (not Gα13)	Localization of Gα12 to lipid rafts	Waheed and Jones, 2002
PP2A	Gα12	Increase in PP2A phosphatase activity	Zhu et al., 2004
PP5	Gα12/13	Gα12/13 interact with TPR domain in PP5, increasing its phosphatase activity	Yamaguchi et al., 2002
Radixin	Gα13	Conformational activation of radixin and its binding to polymerized F-actin	Vaiskunaite et al., 2000
RTK	Gα13 (not Gα12)	Necessary for PDGF-induced cell migration	Shan et al., 2006
Socius	Gα12/13	Gα12/13-dependent RhoA activation	Tateiwa et al., 2005

PP2A, protein phosphatase 2A; PP5, protein phosphatase 5.

In addition to binding to cadherins, Gα₁₂ and Gα₁₃ were found to play important roles in other adhesion molecule-dependent cellular functions. In platelets lacking Gα_q, coactivation of the Gα_q- and Gα_12/13-coupled thromboxane A2 receptor and the Gα_i-coupled ADP receptor P2Y12 resulted in irreversible integrin αIIbβ3-mediated platelet aggregation (Nieswandt et al., 2002). Because Gα_q-deficient platelets fail to respond to thromboxane A2 in αIIbβ3 activation assay, the study demonstrates converging signaling of Gα_12/13 and Gα_i, which is sufficient to overcome Gα_q deficiency for αIIbβ3 activation. A recent study provided evidence for a role of the G₁₂ subfamily in integrin signaling by demonstrating a direct interaction of Gα₁₃ with the cytoplasmic domain of the β3 integrin (Gong et al., 2010). Ligand binding to the integrin αIIbβ3, as well as GTP loading of Gα₁₃, promotes this interaction, supporting an “outside-in” signaling mechanism that favors cell spreading through the activation of c-Src. In platelets, inhibition of Gα₁₃ activation or its expression diminishes the activity of c-Src and augments the activity of RhoA, thus promoting cell retraction (Gong et al., 2010). Therefore, the β3 integrin functions as a noncanonical receptor for Gα₁₃, which is critical to dynamic regulation of c-Src and RhoA.

The above findings are of significance in understanding the role of the G₁₂ subfamily in cell migration, which is relevant to development, host defense, and tumor metastasis. Recent studies have shown that lysophosphatidic acid-induced focal adhesion kinase autophosphorylation is dependent on the Gα_12/13 pathway and contributes to ovarian cancer cell migration (Bian et al., 2006). Likewise, the G₁₂ subfamily was found to be involved in the invasion of prostate cancer cell lines (Kelly et al., 2006). Although agonists for 7TMRs were used in these studies, the role of the G₁₂ subfamily in cell migration may extend further than signaling downstream of 7TMRs. Shan et al. (2006) reported that Gα₁₃-deficient MEFs are defective in cell migration induced by PDGF, a RTK-activating ligand. Of particular interest is the finding that C-terminal deletion mutants of Gα₁₃ unable to couple to 7TMRs can rescue PDGF-induced migration of cells lacking Gα₁₃, suggesting that the RTK-mediated cell migration is Gα₁₃-dependent but independent of its interaction with 7TMRs. In MEFs lacking both Gα₁₂ and Gα₁₃, rescue of cell migration was successful with Gα₁₃ but not Gα₁₂, indicating that the RTK selectively couples to Gα₁₃ for this function (Shan et al., 2006). Recent studies have also shown that the G₁₂ subfamily is important for lymphocyte adhesiveness and motility (Herroeder et al., 2009). In the polarized and migrating promyelocytic leukemic HL-60 cell line, Gα₁₃ and RhoA are localized to the rear of the cell (uropod) and play a role in defining its “backness” (Xu et al., 2003). How Gα₁₃ gets there and what regulates its activation remain unknown. Studies of the integrin outside-in signaling pathways may shed light on the Gα₁₃ regulatory mechanisms, especially if Gα₁₃ can interact with other β integrins. In another study, Gα₁₃ was reported to bind to Hax-1, a cytoskeleton-associated, cortactin-interacting protein. Disruption of this interaction leads to reduced migration of NIH 3T3 cells (Radhika et al., 2004).

The G₁₂ subfamily of G proteins also interacts with phosphatases, including the Ser/Thr protein phosphatase type 5 and protein phosphatase 2A (Yamaguchi et al., 2002; Zhu et al., 2004). In both cases, interaction of the phosphatases with activated Gα₁₂ or Gα₁₃ leads to an increase in the phosphatase activity (Table 2). Although Gα₁₂ and Gα₁₃ sequences are closely related, several studies have highlighted the distinction between Gα₁₂ and Gα₁₃ in their interaction with proteins other than the 7TMRs. For instance, interaction with Hsp90 and protein acylation facilitate Gα₁₂ localization to lipid rafts but have no effect on Gα₁₃ (Waheed and Jones, 2002). This observation also raises the intriguing question of whether Gα₁₂ membrane localization is dependent on Gβγ or other proteins and its post-translational modifications.

Unanswered Questions and Future Perspectives

Mounting evidence for the noncanonical roles of Gα proteins in 7TMR-independent signaling suggests that these G proteins play important physiological functions in addition to coupling to the 7TMRs. For instance, consistent with the finding that Gα₁₃ is pivotal in cell migration, deletion of the Gα₁₃ gene disrupts cell migration necessary for embryonic vasculogenesis in mice, resulting in a lethal phenotype (Offermanns et al., 1997). The recent finding of Gα_i involvement in EGF-stimulated Akt-mTORC1 pathway (Cao et al., 2009) also raises an interesting possibility for the Gα proteins to regulate important developmental processes, because deletion of Gα_i2 and Gα_i3 leads to embryonic lethality as a result of severe growth retardation. Despite the progress made thus far, our understanding of the noncanonical functions of Gα proteins is very limited and far behind what we have learned from investigating G protein coupling to 7TMRs. A number of important questions have not been addressed.

1. It is unclear whether Gα coupling to non-7TMRs is ubiquitous or confined to a selected group of receptors. Cao et al. (2009) found Gα_i1 and Gα_i3 to be critical to EGFR-mediated Akt-mTORC1 activation but not that mediated by insulin receptor, IGFR, and PDGFR, suggesting receptor-dependent selectivity.

2. At present, it is also unclear whether Gβγ proteins play a role in the noncanonical functions of Gα proteins. With the 7TMRs, Gβγ subunits serve the function of anchoring Gα proteins to the plasma membrane, where most of the 7TMRs are located. In addition, Gβγ proteins directly activate downstream effectors including phospholipase C-β, PI3K-γ, the guanine nucleotide exchange factor P-Rex1, and selected ion channels. A number of the non-7TMR Gα-binding proteins are localized intracellularly, raising the question of whether they interact with Gα protein monomers instead of heterotrimers. It is notable that Gβγ-mediated MAP kinase activation involves tyrosine kinase activation and is a pathway shared by both 7TMRs and RTKs (van Biesen et al., 1995). Therefore, Gβγ proteins should be treated separately from Gα proteins because they do not have the intrinsic GTPase activity but are direct activators of several signaling effectors.

3. Based on published data, it is most likely that Gα-mediated noncanonical signaling requires these proteins to enter the activation cycle. However, because most of the published studies were conducted either with constitutively active mutants of Gα₁₂ and Gα₁₃ or with GTP loading, the GEFs and GTPase-activating proteins associated with the noncanonical coupling events remain largely unidentified. In several reported studies in which the G₁₂ family of Gα proteins was found to associate with cytoskeletal proteins and regulate integrin functions, activated Gα₁₂ and/or Gα₁₃ were required. It is presently unclear whether the activated Gα₁₂ and Gα₁₃ proteins are derived from agonist-stimulated 7TMRs or from as-yet-unidentified GEFs. Colocalization of the relevant 7TMRs with these Gα-binding proteins and establishing functional coupling between the noncanonical activation and 7TMR-mediated activation events will shed light on the possible link between 7TMRs and non-7TMR Gα binding proteins. In conclusion, although it is exciting to observe expanded roles of Gα proteins in cell signaling beyond the traditional 7TMRs, much remains to be investigated to appreciate the physiological importance as well as pharmacological mechanisms related to the noncanonical functions of heterotrimeric G proteins.