Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Apr 1;323(Pt 1):239–244. doi: 10.1042/bj3230239

N-terminal binding domain of Galpha subunits: involvement of amino acids 11-14 of Galphao in membrane attachment.

L Busconi 1, P M Boutin 1, B M Denker 1
PMCID: PMC1218301  PMID: 9173888

Abstract

Heterotrimeric guanine nucleotide binding proteins (G-proteins) transmit signals from membrane receptors to a variety of intracellular effectors. G-proteins reversibly associate with components of the signal transduction system, yet remain membrane attached throughout the cycle of activation. The Galpha subunits remain attached to the plasma membrane through a combination of factors that are only partially defined. We now demonstrate that amino acids within the N-terminal domain of Galpha subunits are involved in membrane binding. We used in vitro translation, a technique widely utilized to characterize functional aspects of G-proteins, and interactions with donor-acceptor membranes to demonstrate that amino acids 11-14 of Galphao contribute to membrane binding. The membrane binding of Galphao lacking amino acids 11-14 (D[11-14]) was significantly reduced at all membrane concentrations in comparison with wild-type Galphao. Several other N-terminal mutants of Galphao were characterized as controls, and these results indicate that differences in myristoylation, palmitoylation and betagamma interactions do not account for the reduced membrane binding of D[11-14]. Furthermore, when membrane attachment of Galphao and mutants was characterized in transiently transfected 35S-labelled and [3H]myristate-labelled COS cells, amino acids 11-14 contributed to membrane binding. These studies reveal that membrane binding of Galpha subunits occurs by a combination of factors that include lipids and amino acid sequences. These regions may provide novel sites for interaction with membrane components and allow additional modulation of signal transduction.

Full Text

The Full Text of this article is available as a PDF (294.0 KB).

Selected References

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

  1. Audigier Y., Journot L., Pantaloni C., Bockaert J. The carboxy-terminal domain of Gs alpha is necessary for anchorage of the activated form in the plasma membrane. J Cell Biol. 1990 Oct;111(4):1427–1435. doi: 10.1083/jcb.111.4.1427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature. 1990 Nov 8;348(6297):125–132. doi: 10.1038/348125a0. [DOI] [PubMed] [Google Scholar]
  3. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. doi: 10.1038/349117a0. [DOI] [PubMed] [Google Scholar]
  4. Busconi L., Michel T. Endothelial nitric oxide synthase membrane targeting. Evidence against involvement of a specific myristate receptor. J Biol Chem. 1994 Oct 7;269(40):25016–25020. [PubMed] [Google Scholar]
  5. Cherksey B. D., Zadunaisky J. A., Murphy R. B. Cytoskeletal constraint of the beta-adrenergic receptor in frog erythrocyte membranes. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6401–6405. doi: 10.1073/pnas.77.11.6401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coleman D. E., Berghuis A. M., Lee E., Linder M. E., Gilman A. G., Sprang S. R. Structures of active conformations of Gi alpha 1 and the mechanism of GTP hydrolysis. Science. 1994 Sep 2;265(5177):1405–1412. doi: 10.1126/science.8073283. [DOI] [PubMed] [Google Scholar]
  7. Degtyarev M. Y., Spiegel A. M., Jones T. L. Increased palmitoylation of the Gs protein alpha subunit after activation by the beta-adrenergic receptor or cholera toxin. J Biol Chem. 1993 Nov 15;268(32):23769–23772. [PubMed] [Google Scholar]
  8. Degtyarev M. Y., Spiegel A. M., Jones T. L. Palmitoylation of a G protein alpha i subunit requires membrane localization not myristoylation. J Biol Chem. 1994 Dec 9;269(49):30898–30903. [PubMed] [Google Scholar]
  9. Denker B. M., Boutin P. M., Neer E. J. Interactions between the amino- and carboxyl-terminal regions of G alpha subunits: analysis of mutated G alpha o/G alpha i2 chimeras. Biochemistry. 1995 Apr 25;34(16):5544–5553. doi: 10.1021/bi00016a028. [DOI] [PubMed] [Google Scholar]
  10. Denker B. M., Neer E. J. GO associates with another 40 kDa brain protein. FEBS Lett. 1991 Feb 11;279(1):98–100. doi: 10.1016/0014-5793(91)80260-a. [DOI] [PubMed] [Google Scholar]
  11. Denker B. M., Neer E. J., Schmidt C. J. Mutagenesis of the amino terminus of the alpha subunit of the G protein Go. In vitro characterization of alpha o beta gamma interactions. J Biol Chem. 1992 Mar 25;267(9):6272–6277. [PubMed] [Google Scholar]
  12. Denker B. M., Schmidt C. J., Neer E. J. Promotion of the GTP-liganded state of the Go alpha protein by deletion of the C terminus. J Biol Chem. 1992 May 15;267(14):9998–10002. [PubMed] [Google Scholar]
  13. George D. J., Blackshear P. J. Membrane association of the myristoylated alanine-rich C kinase substrate (MARCKS) protein appears to involve myristate-dependent binding in the absence of a myristoyl protein receptor. J Biol Chem. 1992 Dec 5;267(34):24879–24885. [PubMed] [Google Scholar]
  14. Haun R. S., Tsai S. C., Adamik R., Moss J., Vaughan M. Effect of myristoylation on GTP-dependent binding of ADP-ribosylation factor to Golgi. J Biol Chem. 1993 Apr 5;268(10):7064–7068. [PubMed] [Google Scholar]
  15. Hepler J. R., Biddlecome G. H., Kleuss C., Camp L. A., Hofmann S. L., Ross E. M., Gilman A. G. Functional importance of the amino terminus of Gq alpha. J Biol Chem. 1996 Jan 5;271(1):496–504. doi: 10.1074/jbc.271.1.496. [DOI] [PubMed] [Google Scholar]
  16. Huff R. M., Axton J. M., Neer E. J. Physical and immunological characterization of a guanine nucleotide-binding protein purified from bovine cerebral cortex. J Biol Chem. 1985 Sep 5;260(19):10864–10871. [PubMed] [Google Scholar]
  17. Hunt T. W., Fields T. A., Casey P. J., Peralta E. G. RGS10 is a selective activator of G alpha i GTPase activity. Nature. 1996 Sep 12;383(6596):175–177. doi: 10.1038/383175a0. [DOI] [PubMed] [Google Scholar]
  18. Ibarrondo J., Joubert D., Dufour M. N., Cohen-Solal A., Homburger V., Jard S., Guillon G. Close association of the alpha subunits of Gq and G11 G proteins with actin filaments in WRK1 cells: relation to G protein-mediated phospholipase C activation. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8413–8417. doi: 10.1073/pnas.92.18.8413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Inanobe A., Takahashi K., Katada T. Association of the beta gamma subunits of trimeric GTP-binding proteins with 90-kDa heat shock protein, hsp90. J Biochem. 1994 Mar;115(3):486–492. doi: 10.1093/oxfordjournals.jbchem.a124363. [DOI] [PubMed] [Google Scholar]
  20. Insel P. A., Kennedy M. S. Colchicine potentiates beta-adrenoreceptor-stimulated cyclic AMP in lymphoma cells by an action distal to the receptor. Nature. 1978 Jun 8;273(5662):471–473. doi: 10.1038/273471a0. [DOI] [PubMed] [Google Scholar]
  21. Jahangeer S., Rodbell M. The disaggregation theory of signal transduction revisited: further evidence that G proteins are multimeric and disaggregate to monomers when activated. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):8782–8786. doi: 10.1073/pnas.90.19.8782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Johnson D. R., Bhatnagar R. S., Knoll L. J., Gordon J. I. Genetic and biochemical studies of protein N-myristoylation. Annu Rev Biochem. 1994;63:869–914. doi: 10.1146/annurev.bi.63.070194.004253. [DOI] [PubMed] [Google Scholar]
  23. Jones T. L., Simonds W. F., Merendino J. J., Jr, Brann M. R., Spiegel A. M. Myristoylation of an inhibitory GTP-binding protein alpha subunit is essential for its membrane attachment. Proc Natl Acad Sci U S A. 1990 Jan;87(2):568–572. doi: 10.1073/pnas.87.2.568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Journot L., Pantaloni C., Poul M. A., Mazarguil H., Bockaert J., Audigier Y. Amino acids 367-376 of the Gs alpha subunit induce membrane association when fused to soluble amino-terminal deleted Gi1 alpha subunit. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10054–10058. doi: 10.1073/pnas.88.22.10054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Juhnn Y. S., Jones T. L., Spiegel A. M. Amino- and carboxy-terminal deletion mutants of Gs alpha are localized to the particulate fraction of transfected COS cells. J Cell Biol. 1992 Nov;119(3):523–530. doi: 10.1083/jcb.119.3.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kokame K., Fukada Y., Yoshizawa T., Takao T., Shimonishi Y. Lipid modification at the N terminus of photoreceptor G-protein alpha-subunit. Nature. 1992 Oct 22;359(6397):749–752. doi: 10.1038/359749a0. [DOI] [PubMed] [Google Scholar]
  27. Ladant D. Calcium and membrane binding properties of bovine neurocalcin delta expressed in Escherichia coli. J Biol Chem. 1995 Feb 17;270(7):3179–3185. [PubMed] [Google Scholar]
  28. Lambright D. G., Noel J. P., Hamm H. E., Sigler P. B. Structural determinants for activation of the alpha-subunit of a heterotrimeric G protein. Nature. 1994 Jun 23;369(6482):621–628. doi: 10.1038/369621a0. [DOI] [PubMed] [Google Scholar]
  29. Lambright D. G., Sondek J., Bohm A., Skiba N. P., Hamm H. E., Sigler P. B. The 2.0 A crystal structure of a heterotrimeric G protein. Nature. 1996 Jan 25;379(6563):311–319. doi: 10.1038/379311a0. [DOI] [PubMed] [Google Scholar]
  30. Levis M. J., Bourne H. R. Activation of the alpha subunit of Gs in intact cells alters its abundance, rate of degradation, and membrane avidity. J Cell Biol. 1992 Dec;119(5):1297–1307. doi: 10.1083/jcb.119.5.1297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Linder M. E., Middleton P., Hepler J. R., Taussig R., Gilman A. G., Mumby S. M. Lipid modifications of G proteins: alpha subunits are palmitoylated. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3675–3679. doi: 10.1073/pnas.90.8.3675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Linder M. E., Pang I. H., Duronio R. J., Gordon J. I., Sternweis P. C., Gilman A. G. Lipid modifications of G protein subunits. Myristoylation of Go alpha increases its affinity for beta gamma. J Biol Chem. 1991 Mar 5;266(7):4654–4659. [PubMed] [Google Scholar]
  33. Mixon M. B., Lee E., Coleman D. E., Berghuis A. M., Gilman A. G., Sprang S. R. Tertiary and quaternary structural changes in Gi alpha 1 induced by GTP hydrolysis. Science. 1995 Nov 10;270(5238):954–960. doi: 10.1126/science.270.5238.954. [DOI] [PubMed] [Google Scholar]
  34. Mumby S. M., Heukeroth R. O., Gordon J. I., Gilman A. G. G-protein alpha-subunit expression, myristoylation, and membrane association in COS cells. Proc Natl Acad Sci U S A. 1990 Jan;87(2):728–732. doi: 10.1073/pnas.87.2.728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mumby S. M., Kleuss C., Gilman A. G. Receptor regulation of G-protein palmitoylation. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2800–2804. doi: 10.1073/pnas.91.7.2800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nakamura S., Rodbell M. Octyl glucoside extracts GTP-binding regulatory proteins from rat brain "synaptoneurosomes" as large, polydisperse structures devoid of beta gamma complexes and sensitive to disaggregation by guanine nucleotides. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6413–6417. doi: 10.1073/pnas.87.16.6413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Neer E. J., Denker B. M., Thomas T. C., Schmidt C. J. Analysis of G-protein alpha and beta gamma subunits by in vitro translation. Methods Enzymol. 1994;237:226–239. doi: 10.1016/s0076-6879(94)37065-6. [DOI] [PubMed] [Google Scholar]
  38. Neer E. J. Heterotrimeric G proteins: organizers of transmembrane signals. Cell. 1995 Jan 27;80(2):249–257. doi: 10.1016/0092-8674(95)90407-7. [DOI] [PubMed] [Google Scholar]
  39. Noel J. P., Hamm H. E., Sigler P. B. The 2.2 A crystal structure of transducin-alpha complexed with GTP gamma S. Nature. 1993 Dec 16;366(6456):654–663. doi: 10.1038/366654a0. [DOI] [PubMed] [Google Scholar]
  40. Parenti M., Viganó M. A., Newman C. M., Milligan G., Magee A. I. A novel N-terminal motif for palmitoylation of G-protein alpha subunits. Biochem J. 1993 Apr 15;291(Pt 2):349–353. doi: 10.1042/bj2910349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Popova J. S., Johnson G. L., Rasenick M. M. Chimeric G alpha s/G alpha i2 proteins define domains on G alpha s that interact with tubulin for beta-adrenergic activation of adenylyl cyclase. J Biol Chem. 1994 Aug 26;269(34):21748–21754. [PubMed] [Google Scholar]
  42. Resh M. D. Myristylation and palmitylation of Src family members: the fats of the matter. Cell. 1994 Feb 11;76(3):411–413. doi: 10.1016/0092-8674(94)90104-x. [DOI] [PubMed] [Google Scholar]
  43. Resh M. D. Specific and saturable binding of pp60v-src to plasma membranes: evidence for a myristyl-src receptor. Cell. 1989 Jul 28;58(2):281–286. doi: 10.1016/0092-8674(89)90842-8. [DOI] [PubMed] [Google Scholar]
  44. Sahyoun N. E., LeVine H., 3rd, Davis J., Hebdon G. M., Cuatrecasas P. Molecular complexes involved in the regulation of adenylate cyclase. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6158–6162. doi: 10.1073/pnas.78.10.6158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Silverman L., Resh M. D. Lysine residues form an integral component of a novel NH2-terminal membrane targeting motif for myristylated pp60v-src. J Cell Biol. 1992 Oct;119(2):415–425. doi: 10.1083/jcb.119.2.415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sondek J., Lambright D. G., Noel J. P., Hamm H. E., Sigler P. B. GTPase mechanism of Gproteins from the 1.7-A crystal structure of transducin alpha-GDP-AIF-4. Nature. 1994 Nov 17;372(6503):276–279. doi: 10.1038/372276a0. [DOI] [PubMed] [Google Scholar]
  47. Sternweis P. C. The purified alpha subunits of Go and Gi from bovine brain require beta gamma for association with phospholipid vesicles. J Biol Chem. 1986 Jan 15;261(2):631–637. [PubMed] [Google Scholar]
  48. Taniguchi H., Manenti S. Interaction of myristoylated alanine-rich protein kinase C substrate (MARCKS) with membrane phospholipids. J Biol Chem. 1993 May 15;268(14):9960–9963. [PubMed] [Google Scholar]
  49. Wall M. A., Coleman D. E., Lee E., Iñiguez-Lluhi J. A., Posner B. A., Gilman A. G., Sprang S. R. The structure of the G protein heterotrimer Gi alpha 1 beta 1 gamma 2. Cell. 1995 Dec 15;83(6):1047–1058. doi: 10.1016/0092-8674(95)90220-1. [DOI] [PubMed] [Google Scholar]
  50. Watson N., Linder M. E., Druey K. M., Kehrl J. H., Blumer K. J. RGS family members: GTPase-activating proteins for heterotrimeric G-protein alpha-subunits. Nature. 1996 Sep 12;383(6596):172–175. doi: 10.1038/383172a0. [DOI] [PubMed] [Google Scholar]
  51. Wedegaertner P. B., Bourne H. R. Activation and depalmitoylation of Gs alpha. Cell. 1994 Jul 1;77(7):1063–1070. doi: 10.1016/0092-8674(94)90445-6. [DOI] [PubMed] [Google Scholar]
  52. Wedegaertner P. B., Chu D. H., Wilson P. T., Levis M. J., Bourne H. R. Palmitoylation is required for signaling functions and membrane attachment of Gq alpha and Gs alpha. J Biol Chem. 1993 Nov 25;268(33):25001–25008. [PubMed] [Google Scholar]
  53. Wedegaertner P. B., Wilson P. T., Bourne H. R. Lipid modifications of trimeric G proteins. J Biol Chem. 1995 Jan 13;270(2):503–506. doi: 10.1074/jbc.270.2.503. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES