Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 Aug 1;126(3):811–819. doi: 10.1083/jcb.126.3.811

Localization of a heterotrimeric G protein gamma subunit to focal adhesions and associated stress fibers

PMCID: PMC2120142  PMID: 8045942

Abstract

Signal transducing heterotrimeric G proteins are responsible for coupling a large number of cell surface receptors to the appropriate effector(s). Of the three subunits, 16 alpha, 4 beta, and 5 gamma subunits have been characterized, indicating a potential for over 300 unique combinations of heterotrimeric G proteins. To begin deciphering the unique G protein combinations that couple specific receptors with effectors, we examined the subcellular localization of the gamma subunits. Using anti-peptide antibodies specific for each of the known gamma subunits, neonatal cardiac fibroblasts were screened by standard immunocytochemistry. The anti-gamma 5 subunit antibody yielded a highly distinctive pattern of intensely fluorescent regions near the periphery of the cell that tended to protrude into the cell in a fibrous pattern. Dual staining with anti-vinculin antibody showed co-localization of the gamma 5 subunit with vinculin. In addition, the gamma 5 subunit staining extended a short distance out from the vinculin pattern along the protruding stress fiber, as revealed by double staining with phalloidin. These data indicated that the gamma 5 subunit was localized to areas of focal adhesion. Dual staining of rat aortic smooth muscle cells and Schwann cells also indicated co-localization of the gamma 5 subunit and vinculin, suggesting that the association of the gamma 5 subunit with areas of focal adhesion was wide-spread.

Full Text

The Full Text of this article is available as a PDF (3.5 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arcangeli A., Becchetti A., Mannini A., Mugnai G., De Filippi P., Tarone G., Del Bene M. R., Barletta E., Wanke E., Olivotto M. Integrin-mediated neurite outgrowth in neuroblastoma cells depends on the activation of potassium channels. J Cell Biol. 1993 Sep;122(5):1131–1143. doi: 10.1083/jcb.122.5.1131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnbaumer L. Receptor-to-effector signaling through G proteins: roles for beta gamma dimers as well as alpha subunits. Cell. 1992 Dec 24;71(7):1069–1072. doi: 10.1016/s0092-8674(05)80056-x. [DOI] [PubMed] [Google Scholar]
  3. Blank J. L., Brattain K. A., Exton J. H. Activation of cytosolic phosphoinositide phospholipase C by G-protein beta gamma subunits. J Biol Chem. 1992 Nov 15;267(32):23069–23075. [PubMed] [Google Scholar]
  4. Burridge K., Turner C. E., Romer L. H. Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J Cell Biol. 1992 Nov;119(4):893–903. doi: 10.1083/jcb.119.4.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cali J. J., Balcueva E. A., Rybalkin I., Robishaw J. D. Selective tissue distribution of G protein gamma subunits, including a new form of the gamma subunits identified by cDNA cloning. J Biol Chem. 1992 Nov 25;267(33):24023–24027. [PubMed] [Google Scholar]
  6. Camps M., Hou C., Sidiropoulos D., Stock J. B., Jakobs K. H., Gierschik P. Stimulation of phospholipase C by guanine-nucleotide-binding protein beta gamma subunits. Eur J Biochem. 1992 Jun 15;206(3):821–831. doi: 10.1111/j.1432-1033.1992.tb16990.x. [DOI] [PubMed] [Google Scholar]
  7. Carey D. J., Stahl R. C., Cizmeci-Smith G., Asundi V. K. Syndecan-1 expressed in Schwann cells causes morphological transformation and cytoskeletal reorganization and associates with actin during cell spreading. J Cell Biol. 1994 Jan;124(1-2):161–170. doi: 10.1083/jcb.124.1.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carey D. J., Stahl R. C. Identification of a lipid-anchored heparan sulfate proteoglycan in Schwann cells. J Cell Biol. 1990 Nov;111(5 Pt 1):2053–2062. doi: 10.1083/jcb.111.5.2053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Conklin B. R., Bourne H. R. Structural elements of G alpha subunits that interact with G beta gamma, receptors, and effectors. Cell. 1993 May 21;73(4):631–641. doi: 10.1016/0092-8674(93)90245-l. [DOI] [PubMed] [Google Scholar]
  10. Crawford A. W., Michelsen J. W., Beckerle M. C. An interaction between zyxin and alpha-actinin. J Cell Biol. 1992 Mar;116(6):1381–1393. doi: 10.1083/jcb.116.6.1381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Federman A. D., Conklin B. R., Schrader K. A., Reed R. R., Bourne H. R. Hormonal stimulation of adenylyl cyclase through Gi-protein beta gamma subunits. Nature. 1992 Mar 12;356(6365):159–161. doi: 10.1038/356159a0. [DOI] [PubMed] [Google Scholar]
  12. Fisher K. J., Aronson N. N., Jr Characterization of the cDNA and genomic sequence of a G protein gamma subunit (gamma 5). Mol Cell Biol. 1992 Apr;12(4):1585–1591. doi: 10.1128/mcb.12.4.1585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Foster K. A., McDermott P. J., Robishaw J. D. Expression of G proteins in rat cardiac myocytes: effect of KCl depolarization. Am J Physiol. 1990 Aug;259(2 Pt 2):H432–H441. doi: 10.1152/ajpheart.1990.259.2.H432. [DOI] [PubMed] [Google Scholar]
  14. Gautam N., Northup J., Tamir H., Simon M. I. G protein diversity is increased by associations with a variety of gamma subunits. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7973–7977. doi: 10.1073/pnas.87.20.7973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gilman A. G. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615–649. doi: 10.1146/annurev.bi.56.070187.003151. [DOI] [PubMed] [Google Scholar]
  16. Haga K., Haga T. Activation by G protein beta gamma subunits of agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptors and rhodopsin. J Biol Chem. 1992 Feb 5;267(4):2222–2227. [PubMed] [Google Scholar]
  17. Hamati H. F., Britton E. L., Carey D. J. Inhibition of proteoglycan synthesis alters extracellular matrix deposition, proliferation, and cytoskeletal organization of rat aortic smooth muscle cells in culture. J Cell Biol. 1989 Jun;108(6):2495–2505. doi: 10.1083/jcb.108.6.2495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Horiuchi H., Kaibuchi K., Kawamura M., Matsuura Y., Suzuki N., Kuroda Y., Kataoka T., Takai Y. The posttranslational processing of ras p21 is critical for its stimulation of yeast adenylate cyclase. Mol Cell Biol. 1992 Oct;12(10):4515–4520. doi: 10.1128/mcb.12.10.4515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hurley J. B., Fong H. K., Teplow D. B., Dreyer W. J., Simon M. I. Isolation and characterization of a cDNA clone for the gamma subunit of bovine retinal transducin. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6948–6952. doi: 10.1073/pnas.81.22.6948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hynes R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. doi: 10.1016/0092-8674(92)90115-s. [DOI] [PubMed] [Google Scholar]
  21. Inglese J., Koch W. J., Caron M. G., Lefkowitz R. J. Isoprenylation in regulation of signal transduction by G-protein-coupled receptor kinases. Nature. 1992 Sep 10;359(6391):147–150. doi: 10.1038/359147a0. [DOI] [PubMed] [Google Scholar]
  22. Iñiguez-Lluhi J. A., Simon M. I., Robishaw J. D., Gilman A. G. G protein beta gamma subunits synthesized in Sf9 cells. Functional characterization and the significance of prenylation of gamma. J Biol Chem. 1992 Nov 15;267(32):23409–23417. [PubMed] [Google Scholar]
  23. Kim D., Lewis D. L., Graziadei L., Neer E. J., Bar-Sagi D., Clapham D. E. G-protein beta gamma-subunits activate the cardiac muscarinic K+-channel via phospholipase A2. Nature. 1989 Feb 9;337(6207):557–560. doi: 10.1038/337557a0. [DOI] [PubMed] [Google Scholar]
  24. Kleuss C., Scherübl H., Hescheler J., Schultz G., Wittig B. Different beta-subunits determine G-protein interaction with transmembrane receptors. Nature. 1992 Jul 30;358(6385):424–426. doi: 10.1038/358424a0. [DOI] [PubMed] [Google Scholar]
  25. Kleuss C., Scherübl H., Hescheler J., Schultz G., Wittig B. Selectivity in signal transduction determined by gamma subunits of heterotrimeric G proteins. Science. 1993 Feb 5;259(5096):832–834. doi: 10.1126/science.8094261. [DOI] [PubMed] [Google Scholar]
  26. Lerea C. L., Somers D. E., Hurley J. B., Klock I. B., Bunt-Milam A. H. Identification of specific transducin alpha subunits in retinal rod and cone photoreceptors. Science. 1986 Oct 3;234(4772):77–80. doi: 10.1126/science.3529395. [DOI] [PubMed] [Google Scholar]
  27. Lotersztajn S., Pavoine C., Deterre P., Capeau J., Mallat A., LeNguyen D., Dufour M., Rouot B., Bataille D., Pecker F. Role of G protein beta gamma subunits in the regulation of the plasma membrane Ca2+ pump. J Biol Chem. 1992 Feb 5;267(4):2375–2379. [PubMed] [Google Scholar]
  28. Maltese W. A., Robishaw J. D. Isoprenylation of C-terminal cysteine in a G-protein gamma subunit. J Biol Chem. 1990 Oct 25;265(30):18071–18074. [PubMed] [Google Scholar]
  29. Marshall C. J. Protein prenylation: a mediator of protein-protein interactions. Science. 1993 Mar 26;259(5103):1865–1866. doi: 10.1126/science.8456312. [DOI] [PubMed] [Google Scholar]
  30. Mumby S. M., Casey P. J., Gilman A. G., Gutowski S., Sternweis P. C. G protein gamma subunits contain a 20-carbon isoprenoid. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5873–5877. doi: 10.1073/pnas.87.15.5873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Musha T., Kawata M., Takai Y. The geranylgeranyl moiety but not the methyl moiety of the smg-25A/rab3A protein is essential for the interactions with membrane and its inhibitory GDP/GTP exchange protein. J Biol Chem. 1992 May 15;267(14):9821–9825. [PubMed] [Google Scholar]
  32. Peng Y. W., Robishaw J. D., Levine M. A., Yau K. W. Retinal rods and cones have distinct G protein beta and gamma subunits. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10882–10886. doi: 10.1073/pnas.89.22.10882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Phillips W. J., Cerione R. A. Rhodopsin/transducin interactions. I. Characterization of the binding of the transducin-beta gamma subunit complex to rhodopsin using fluorescence spectroscopy. J Biol Chem. 1992 Aug 25;267(24):17032–17039. [PubMed] [Google Scholar]
  34. Phillips W. J., Wong S. C., Cerione R. A. Rhodopsin/transducin interactions. II. Influence of the transducin-beta gamma subunit complex on the coupling of the transducin-alpha subunit to rhodopsin. J Biol Chem. 1992 Aug 25;267(24):17040–17046. [PubMed] [Google Scholar]
  35. Pitcher J. A., Inglese J., Higgins J. B., Arriza J. L., Casey P. J., Kim C., Benovic J. L., Kwatra M. M., Caron M. G., Lefkowitz R. J. Role of beta gamma subunits of G proteins in targeting the beta-adrenergic receptor kinase to membrane-bound receptors. Science. 1992 Aug 28;257(5074):1264–1267. doi: 10.1126/science.1325672. [DOI] [PubMed] [Google Scholar]
  36. Pronin A. N., Gautam N. Interaction between G-protein beta and gamma subunit types is selective. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6220–6224. doi: 10.1073/pnas.89.13.6220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Robishaw J. D., Balcueva E. A. A high temperature transfer procedure for detection of G protein gamma subunits by immunoblotting. Anal Biochem. 1993 Feb 1;208(2):283–287. doi: 10.1006/abio.1993.1047. [DOI] [PubMed] [Google Scholar]
  38. Robishaw J. D., Balcueva E. A. Preparation, characterization, and use of antibodies with specificity for G-protein gamma subunits. Methods Enzymol. 1994;237:498–509. doi: 10.1016/s0076-6879(94)37086-9. [DOI] [PubMed] [Google Scholar]
  39. Robishaw J. D., Foster K. A. Role of G proteins in the regulation of the cardiovascular system. Annu Rev Physiol. 1989;51:229–244. doi: 10.1146/annurev.ph.51.030189.001305. [DOI] [PubMed] [Google Scholar]
  40. Robishaw J. D., Kalman V. K., Moomaw C. R., Slaughter C. A. Existence of two gamma subunits of the G proteins in brain. J Biol Chem. 1989 Sep 25;264(27):15758–15761. [PubMed] [Google Scholar]
  41. Robishaw J. D., Kalman V. K., Proulx K. L. Production, processing and partial purification of functional G protein beta gamma subunits in baculovirus-infected insect cells. Biochem J. 1992 Sep 15;286(Pt 3):677–680. doi: 10.1042/bj2860677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schaller M. D., Borgman C. A., Cobb B. S., Vines R. R., Reynolds A. B., Parsons J. T. pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5192–5196. doi: 10.1073/pnas.89.11.5192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schmidt C. J., Thomas T. C., Levine M. A., Neer E. J. Specificity of G protein beta and gamma subunit interactions. J Biol Chem. 1992 Jul 15;267(20):13807–13810. [PubMed] [Google Scholar]
  44. Simon M. I., Strathmann M. P., Gautam N. Diversity of G proteins in signal transduction. Science. 1991 May 10;252(5007):802–808. doi: 10.1126/science.1902986. [DOI] [PubMed] [Google Scholar]
  45. Smrcka A. V., Sternweis P. C. Regulation of purified subtypes of phosphatidylinositol-specific phospholipase C beta by G protein alpha and beta gamma subunits. J Biol Chem. 1993 May 5;268(13):9667–9674. [PubMed] [Google Scholar]
  46. Tang W. J., Gilman A. G. Type-specific regulation of adenylyl cyclase by G protein beta gamma subunits. Science. 1991 Dec 6;254(5037):1500–1503. doi: 10.1126/science.1962211. [DOI] [PubMed] [Google Scholar]
  47. Yamane H. K., Farnsworth C. C., Xie H. Y., Howald W., Fung B. K., Clarke S., Gelb M. H., Glomset J. A. Brain G protein gamma subunits contain an all-trans-geranylgeranylcysteine methyl ester at their carboxyl termini. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5868–5872. doi: 10.1073/pnas.87.15.5868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zachary I., Rozengurt E. Focal adhesion kinase (p125FAK): a point of convergence in the action of neuropeptides, integrins, and oncogenes. Cell. 1992 Dec 11;71(6):891–894. doi: 10.1016/0092-8674(92)90385-p. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES