Gene symbols: GNA12, GNA13

1. General Information

Gα_12/13 are the unique α subunits of a class of heterotrimeric G proteins along with Gα_S, Gα_i/o, and Gα_q.  α₁₂ and α₁₃ were initially cloned from a mouse brain cDNA library by PCR and show 67% amino acid identity with each other but only 35-40% with other Gα subunits (32).  These α subunits are expressed in most tissues (34) and are activated by over 25 receptors mostly of the 7TM class but also by some receptor tyrosine kinases (18,27).  A Drosophila homolog known as α_cta is 55% identical at the amino acid level.  The Gα_12/13 polypeptides have a Mol Wt of 43,000 and are not ribosylated by Pertussis or Cholera toxin.  They are palmitoylated at a cysteine residue near the N-terminal which is believed important for plasma membrane targeting (1);  Gα₁₂ but not Gα₁₃ is located predominately in lipid rafts (37).  This targeting also depends upon interaction with HSP90.  In addition, the biochemical properties of Gα₁₂ can be modified by phosphorylation and PKC phosphorylates purified α₁₂ (18).  Not surprisingly because of their divergence in sequence, α₁₂ and α₁₃ do not always show the same effect (2,13) and are not always activated by the same agonist (24).  This difference is mediated by a short N terminal sequence where homology is only 16% (39).  In addition to these differences, in several cell types Gα₁₂ and Gα₁₃ have shown different subcellular localization with Gα₁₂ localized to the plasma membrane while Gα₁₃ localizes to the cyosol and upon stimulation translocates to the plasma membrane (40).  A striking difference is that Gα₁₂ deficient mice are viable with no obvious phenotype while Gα₁₃ deficient mice die in mid-gestation with defects in angiogenesis (26).  This defect is due to an essential role for Gα₁₃ in endothelial cells (28).

As with other G proteins, G_12/13 undergoes a cycle where receptor induced activation involves binding of GTP which is latter hydrolyzed to GDP returning the protein to the inactive state.  These changes are mediated by GEFs and GAPs with the receptor acting as a GEF when liganded.  Several RGS proteins particularly RGS1 and -16 may also regulate G_12/13 (13).  Constitutively active forms (Gα₁₂ Q229L or Gα₁₃ Q226L) can induce transformation of fibroblasts and tumorigenesis in animal models.  Gα₁₂ has a role in cell-cell interactions, invasion and differentiation (14).  In addition to stimulating DNA synthesis, active Gα_12/13 promotes stress fibers and cell adhesion, inhibits cadherin-induced aggregation, activates or inhibits Na⁺- H⁺ exchange, stimulates smooth muscle contraction, and affects secretion (11,13,22). Tissue specific knockout studies have shown the requirement for Gα12/13 for developmental cell migration in the brain (21).  Gα_12/13 also plays a role in platelet activation, cardiovascular function and immune function (25,38).

Use of constitutively active mutants of α₁₂ and α₁₃ has generally shown that these G proteins do not regulate adenylate cyclase or phospholipase C ( but see Ref 10 for an exception).  The most well studied action of Gα_12/13 is to activate the small G protein Rho in response to a GPCR and through RhoA and its downstream effectors affect the actin cytoskeleton, cell migration and invasion, phospholipase D activation, protein kinase D activation, Na⁺-H⁺ exchange, JNK activation and serum response factor (SRF) production (4,7,13,15,22,27,30,33).  All these actions are sensitive to C. botulinum C3 exotoxin which inactivates Rho.  More recently, Rho activation has been directly measured through pull down assays.  The primary mechanism for Gα_12/13 to activate Rho involves Rho GEFS which contain a RGS like domain that binds to active α₁₂ or α₁₃ (31).  Three Rho GEFs have been identified with RGS like domains near the amino terminus, p115 RhoGEF, PDZ-RhoGEF and leukemia associated RhoGEF (LARG) (6,35).  Another RhoGEF, Lbc-RhoGEF has a RGS like domain of lower homology in the carboxyl terminus and is activated selectively by Gα₁₂ (5).  All of these RhoGEFs are potent RhoA activators while the isolated RGS like domain from p115 RhoGEF when overexpressed acts as a specific inhibitor of G_12/13 signaling through Rho.  In some cases for full RhoGEF activation the GEF also has to be phosphorylated by a nonreceptor tyrosine kinase.  Gα₁₃ also activates a RhoGEF without a RGS like domain, proto-Dbl which translocates to the plasma membrane where it interacts with ezrin (36).  In addition to these RhoGEFs, activated Gα_12/13 can also bind to certain cadherins, the protein radixin of the ERM family, some nonreceptor tyrosine kinases (Bruton’s tyrosine kinase or BTK and Tec) , some AKAPS, zonula occludens proteins, protein phosphatase type 5  and JNK-interacting protein (JIP) (12,13,17).  Thus in some cell types active Gα₁₂ or Gα₁₃ can activate signaling independent of Rho.  These include activation of JNK, ERKs, Pyk2, and phospholipase A2.  For example, in a thyroid cell line, Gα₁₃ but not Gα₁₂ activated ERK and subsequent induction of c-Fos independent of Rho (3).

2. Specific function in the pancreas

Only a few studies have addressed the role of Gα_12/13 in pancreatic cells.  Both α₁₂ and α₁₃ were reported to be present in rat pancreatic acini as shown by Western blotting (16).  In this study CCK was shown to rapidly increase the expression of both α12 and α13 as well as increasing the association of RhoA and Vav2 with Gα₁₃ but not Gα₁₂.  In mouse pancreas and pancreatic acini  both PCR and Western blotting revealed the presence of Gα₁₃ but not Gα₁₂ (29).  In accord with previous studies (1), Gα₁₃ was associated with a membrane fraction in both control and stimulated acini.

Constitutively active Gα₁₃ (Q226L) delivered by adenoviral vector was shown to activate RhoA similar to CCK in mouse acini and to alter the actin cytoskeleton leading to bleb formation (29).  In this study, expression of a p115 RhoGEF RGS like domain (p115-RGS) abolished RhoA activation in response to CCK suggesting that the action of CCK receptors to activate RhoA was mediated by Gα_12/13.  Similar results had been reported earlier in intestinal smooth muscle cells expressing Gα13 (23) and in NIH 3T3 cells stabely transfected with CCKA receptors (19).  A mutant form of p115-RGS (E29K) failed to modify CCK-induced RhoA activation (29).  The effect of p115-RGS expression was shown to be specific for G_12/13 signaling as it had no effect on Ca²⁺ mobilization or cAMP formation.  Expression of p115-RGS inhibited both basal and CCK-stimulated amylase release.  Prior studies had shown that inhibition of RhoA activation by C3 exotoxin or dominant negative RhoA also inhibited amylase release (2).  Thus these results suggest that CCK-induced activation of Gα₁₃ in addition to CCK-induced activation of  Gα_q/11  is responsible for induction of amylase secretion.  Whereas activation of Gα_q stimulates PLC and calcium mobilization, activation of Gα₁₃ induces activation of RhoA and reorganization of the actin cytoskeleton.  At present the nature of the specific RhoGEF activated in acinar cells by Gα₁₃ is unknown.  Both p115 RhoGEF and LARG have been identified by PCR in acinar cells (unpublished data).  Whether Gα_12/13 will have other actions in acinar cells or plays a role in other pancreatic cells such as pancreatic stellate cells remains to be determined. The position of Gα₁₃ in the pathway of Rho activation in mouse acinar cells is shown in the schema below.  For further details see Gα_12/13 – RhoA in the Pathways section

3. Tools to study Gα_12/13

a. cDNA Clones

Multiple clones for Gα_12/13 are available from Missouri Science and Techology cDNA resource.  A number of investigators have prepared or used plasmids for Gα12/13 and their constitutively active mutants, Gα₁₂ Q229L and Gα₁₃ Q226L.  Dominant negative mutants have also been prepared, Gα₁₂ G228A and Gα₁₃ G225A and shown to block stress fiber formation (8).

b. Antibodies 

We have used antibodies from Santa Cruz to Gα₁₂ (sc-409) and Gα₁₃ (sc-410) and a rabbit polyclonal to Gα₁₂ from Abcam (ab35016) for Western blotting in our studies of mouse pancreas (29).  Other commercial antibodies to Gα₁₂ are listed in Ref (18).  A series of antibodies against peptide sequences and validation of their specificity was also carried out by the group of G. Schultz (34).

c. Viral Vectors 

Constitutively active Gα₁₃ Q226L in a adenoviral vector has been prepared and used by us in mouse pancreatic acini (29).

d. Mouse lines

Gene deletion has been carried out for Gna13 where mice died around embryonic day 10 (26) and for Gna12 where mice develop normally (9).  Mice with floxed Gna13 have been generated and used for tissue specific deletion with Cre alone or combined with Gna12 deletion (20,21).

4. References

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2. Bi Y, and Williams JA.  A role for Rho and Rac in secretagogue-induced amylase release by pancreatic acini.  Am J Physiol Cell Physiol.  289:C22-C32, 2005. PMID: 15743890
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