Galpha 12/13

Department of Molecular and Integrative Physiology, The University of Michigan, Ann Arbor, Michigan 48109-0622

Entry Version: 

Version 1.0, March 1, 2011


Williams, John A. (2011). Galpha12/13.
Pancreapedia: Exocrine Pancreas Knowledge Base, DOI: 10.3998/panc.2011.7
PDF icon Galpha 12/13254.62 KB

Gene symbols: GNA12, GNA13

1. General Information

12/13 are the unique α subunits of a class of heterotrimeric G proteins along with GαS, Gαi/o, and Gαq.  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 αcta is 55% identical at the amino acid level.  The Gα12/13 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, G12/13 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 G12/13 (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 Gα12/13 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).  Gα12/13 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 Gα12/13 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 Gα12/13 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 G12/13 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 Gα12/13 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 Gα12/13 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 Gα12/13.  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 G12/13 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  Gαq/11  is responsible for induction of amylase secretion.  Whereas activation of Gαq 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 Gα12/13 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 Gα12/13 – RhoA in the Pathways section

3. Tools to study Gα12/13

a. cDNA Clones

Multiple clones for Gα12/13 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

  1. Bhattacharyya R, and Wedegaertner PB. 13 requires palmitoylation for plasma membrane localization, Rho-dependent signaling, and promotion of p115-RhoGEF membrane binding.  J Biol Chem  275:14992-14999, 2000. PMID: 10747909
  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
  3. Buch TRH, Biebermann H, Kalwa H, Pinkenberg O, Hager D, Barth H, Aktories K, Breit A, and Gudermann T.  G13-dependent activation of MAPK by thyrotropin.  J Biol Chem.  283:20330-20341, 2008. PMID: 18445595
  4. Buhl AM, Johnson NL, Dhanasekaran N, and Johnson GI.  Gα12 and Gα13 stimulate Rho-dependent stress fiber formation and focal adhesion assembly.  J Biol Chem.  270:24631-24634, 1995. PMID: 7559569
  5. Dutt P, Nguyen N, and Toksoz D.  Role of Lbc RhoGEF in Gα12/13-induced signals to Rho GTPase.  Cellular Signalling.  16:201-209, 2004. PMID: 14636890
  6. Fukuhara S, Chikumi H and Gutkind JS.  RGS-containing RhoGEFs: the missing link between transforming G proteins and Rho? Oncogene, 20:1661-1668, 2001. PMID: 11313914
  7. Gavard J, and Gutkind JS. Protein kinase C-related kinase and ROCK are required for thrombin-induced endothelial cell permeability downstream from Galpha12/13 and Galpha11/q.  J Biol Chem.  283:29888-29896, 2008.  PMID: 18713748
  8. Gohla A, Offermanns S, Wilkie TM, and Schultz G.  Differential involvement of Gα12 and Gα13 in receptor-mediated stress fiber formation.  J Biol Chem. 274:17901-17907, 1999. PMID: 10364236
  9. Gu JL, Muller S, Mancino V, Offermanns S, and Simon MI.  Interaction of Gα12 with Gα13 and Gαq signaling pathways.  Proc Natl Acad Sci USA  99:9352-9357, 2002. PMID: 12077299
  10. Jiang LI, Collins J, Davis R, Fraser ID and Sternweis PC.  Regulation of cAMP responses by the G12/13 pathway converges on adenyl cyclase VII. J Biol Chem. 283:23429-23439, 2008. PMID: 18541530
  11. Juneja J and Casey PJ.  Role of G12 proteins in oncogenesis and metastasis.  Br J Pharmacol.  158:32-40, 2009. PMID: 19422395
  12. Kashef K, Lee CM, Ha JH, Reddy P, and Dhanasekaran DN.  JNK-interacting leucine zipper protein is a novel scaffolding protein in the Gα13 signaling pathway.  Biochemistry  44:14090-14096, 2005. PMID: 16245925
  13. Kelly P, Casey PJ and Meigs TE.  Biologic functions of the G12 subfamily of heterotrimeric G proteins: growth, migration, and metastasis.  Biochemistry 46:6677-6687, 2007. PMID: 17503779
  14. Kelly P, Moeller BJ, Juneja J, Booden MA, Der CJ, Daaka Y, Dewhirst MW, Fields TA, and Casey PJ.  The G12 family of heterotrimeric G proteins promotes breast cancer invasion and metastasis.  Proc Natl Acad Sci USA.  103:8173-8178. PMID: 16705036
  15. Kelly P, Stemmle LN, Madden JF, Fields TA, Daaka Y, and Casey PJ.  A role for the G12 family of hererotrimeric G proteins in prostate cancer invasion.  J Biol Chem.  281:26483-26490, 2006.  PMID: 16787920
  16. Kim M, Nozu F, Kusama K and Imawari M.  Cholecystokinin stimulates the recruitment of the Src-RhoA-phosphoinositide 3-kinase pathway by Vav-2 downstream of Gα13 in pancreatic acini.  Biochem Biophys Res Commun.  339:271-276, 2006. PMID: 16297869
  17. Kurose H.  Gα12 and Gα13 as key regulatory mediator in signal transduction.  Life Sciences 74:155-161, 2003. PMID: 14607242
  18. Lee WH, Lee CH, Moon A, Dhanasekaran DN and Kim SG.  G protein alpha 12.  Nature Molecule Pages Published online: 25 Mar 2010 l doi:10.1038/mp.a000039.01, 2010.
  19. LePage SL, Bi Y, and Williams JA.  CCK-A receptor activates RhoA through Gα12/13 in NIH3T3 cells.  Am J Physiol Cell Physiol.  285:C1197-C1206, 2003. PMID: 12853286
  20. Moers A, Nieswandt B, Massberg S, Wettschureck N, Gruner S, Konrad I, Schulte V, Aktas B, Gratacap M-P, Simon MI, Gawaz M, and Offermanns S.  G13 is an essential mediator of platelet activation in hemostasis and thrombosis.  Nature Medicine  9:1418-1422, 2003. PMID: 14528298
  21. Moers A, Nurnberg A, Goebbels S, Wettschureck N and Offermanns S.  Gα12/Gα13 deficiency causes localized overmigration of neurons in the developing cerebral and cerebellar cortices.  Mol Cell Biol 28:1480-1488, 2008. PMID: 18086886
  22. Murthy KS. Signaling for contraction and relaxation in smooth muscle of the gut.  Annual Review of Physiol. 68:345-374, 2006. PMID: 16460276
  23. Murthy KS, Zhou H, Grider JR, and Makhlouf GM.  Sequential activation of heterotrimeric and monomeric G proteins mediates PLD activity in smooth muscle.  Am J Physiol Gastrointest Liver Physiol.  280:G381-G388, 2001.  PMID: 11171620
  24. Nürnberg A, Braüer AU, Wettschureck N, and Offermans S.  Antagonistic regulation of neurite morphology through Gq/G11 and G12/G13.  J Biol Chem.  283:35526-35531, 2008. PMID: 18854320.
  25. Offermans S.  In vivo functions of heterotrimeric G-proteins: studies in Gα-deficient mice.  Oncogene 20:1635-1642, 2001. PMID: 11313911
  26. Offermanns S, Mancino V, Revel J-P, and Simon MI.  Vascular system defects and impaired cell chemokinesis as a result of Gα13 deficiency.  Science 275:533-536, 1997. PMID: 8999798
  27. Riobo NA and Manning DR.  Receptors coupled to heterotrimeric G proteins of the G12 family.  Trends in Pharmacological Sciences.  26:146-154, 2005. PMID: 15749160
  28. Ruppel KM, Willison D, Kataoka H, Wang A, Zheng Y-W, Cornelissen I, Yin L, Xu SM, and Coughlin SR.  Essential role for Gα13 in endothelial cells during embryonic development.  Proc Natl Acad Sci USA,  102:8281-8286. PMID: 15919816
  29. Sabbatini ME, Bi Y, Ji B, Ernst SA and Williams JA.  CCK activates RhoA and Rac1 differentially through Gα13 and Gαq in mouse pancreatic acini.  Am J Physiol Cell Physiol. 298:C592-C601, 2010. PMID: 19940064
  30. Sah VP, Seasholtz TM, Sagi, SA, and Brown JH.  The role of Rho in G protein-coupled receptor signal transduction.  Annu Rev Pharmacol Toxicol.  40:459-489, 2000. PMID: 10836144
  31. Siehler S.  Regulation of RhoGEF proteins by G12/13-coupled receptors.  Br J Pharmacol. 158:41-49, 2009. PMID: 19226283
  32. Simon MI, Strathmann MP, Gautam N.  Diversity of G proteins in signal transduction.  Science  252:802-808, 1991. PMID: 1902986
  33. Simoncini T, Scorticati C, Mannella P, Fadiel A, Giretti MS, Fu X-D, Baldacci C, Garibaldi S, Caruso A, Fornari L, Naftolin F, and Genazzani AR. Estrogen Receptor α interacts with Gα13 to drive actin remodeling and endothelial cell migration via the RhoA/Rho kinas/moesin pathway.  Mol Endocrinol.  20:1756-1771, 2006. PMID: 16601072
  34. Spicher K, Kalkbrenner F, Zobel A, Harhammer R, Nürnberg B, Söling A, and Schultz G.   G12 and G13 α-subunits are immunochemically detectable in most membranes of various mammalian cells and tissues.  Biochem Biophys Res Commun. 198:906-914, 1994. PMID: 8117295
  35. Sternweis P, Carter AM, Chen Z, Danesh SM, Hsiung YF and Singer WD.  Regulation of Rho guanine nucleotide exchange factors by G proteins.  Adv Protein Chem.  74:189-228, 2007. PMID: 17854659
  36. Vanni C, Mancini P, Ottaviano C, Ognibene M, Parodi A, Merello E, Russo C, Varesio L, Zheng Y, Torrisi MR, and Eva A.  Gα13 regulation of proto-Dbl signaling.  Cell Cycle 6:2058-2070, 2007. PMID: 17721084
  37. Waheed AA and Jones TLZ. Hsp90 interactions and acylation target the Gprotein Gα12 but not Gα13 to lipid rafts.  J Biol Chem.  277:32409-32412, 2002. PMID: 12117999
  38. Worzfeld T, Wettschureck N, and Offermanns S.  G12/G13-mediated signaling in mammalian physiology and disease.  Trends in Pharmacol Sci.  29:582-589, 2008. PMID: 18814923
  39. Yamaguchi Y, Katoh H, and Negishi M.  N-terminal short sequences of α subunits of the G12 family determine selective coupling to receptors.  J Biol Chem.  278:14936-14939, 2003. PMID: 12594220
  40. Yamazaki J, Katoh H, Yamaguchi Y, and Negishi M.  Two G12 family G proteins, Gα12 and Gα13 show different subcellular localization.  Biochem Biophys Res Commun 332:782-786, 2005. PMID: 15907792