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. 1998 Feb 15;330(Pt 1):287–293. doi: 10.1042/bj3300287

Functional rescue of a constitutively desensitized beta2AR through receptor dimerization.

T E Hebert 1, T P Loisel 1, L Adam 1, N Ethier 1, S S Onge 1, M Bouvier 1
PMCID: PMC1219139  PMID: 9461522

Abstract

We have recently demonstrated that wild-type beta2-adrenergic receptors (beta2AR) form homodimers and that disruption of receptor dimerization inhibits signalling via Gs [Hebert, Moffett, Morello, Loisel, Bichet, Barret and Bouvier (1996) J. Biol. Chem. 271, 16384-16392]. Here taking advantage of the altered functional properties of a non-palmitoylated, constitutively desensitized mutant beta2AR (C341Gbeta2AR), we sought to study whether physical interactions between mutant and wild-type beta2AR expressed in Sf9 cells could occur and have functional consequences. Using metabolic labelling with [3H]palmitate and co-immunoprecipitation we demonstrated the existence of heterodimerization between wild-type and C341Gbeta2AR. Furthermore, we show that, in co-expression experiments, wild-type receptors have a dominant positive effect resulting in the functional complementation of C341Gbeta2AR. Indeed, when expressed alone, the mutant C341G receptor displays altered functional characteristics in that (1) the response of the receptor to agonist is reduced as compared to the wild-type receptor and (2) the desensitization of the receptor in response to prolonged exposure to agonist is minimal. In contrast, when C341G and the wild-type beta2AR were expressed together, both the response to agonist and subsequent desensitization (at a constant level of total receptor) were equivalent to the wild-type beta2AR expressed alone. This dominant positive effect was also seen when C341G was co-expressed with a second receptor mutant in which the two protein kinase A phosphorylation sites (S261, 262, 345, 346A beta2AR) were mutated. Taken together these data suggest that intermolecular interactions between receptors may have both functional and structural implications for G-protein-mediated signalling.

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Selected References

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  1. Avissar S., Amitai G., Sokolovsky M. Oligomeric structure of muscarinic receptors is shown by photoaffinity labeling: subunit assembly may explain high- and low-affinity agonist states. Proc Natl Acad Sci U S A. 1983 Jan;80(1):156–159. doi: 10.1073/pnas.80.1.156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benovic J. L., Bouvier M., Caron M. G., Lefkowitz R. J. Regulation of adenylyl cyclase-coupled beta-adrenergic receptors. Annu Rev Cell Biol. 1988;4:405–428. doi: 10.1146/annurev.cb.04.110188.002201. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bouvier M., Chidiac P., Hebert T. E., Loisel T. P., Moffett S., Mouillac B. Dynamic palmitoylation of G-protein-coupled receptors in eukaryotic cells. Methods Enzymol. 1995;250:300–314. doi: 10.1016/0076-6879(95)50080-4. [DOI] [PubMed] [Google Scholar]
  5. Bouvier M., Hausdorff W. P., De Blasi A., O'Dowd B. F., Kobilka B. K., Caron M. G., Lefkowitz R. J. Removal of phosphorylation sites from the beta 2-adrenergic receptor delays onset of agonist-promoted desensitization. Nature. 1988 May 26;333(6171):370–373. doi: 10.1038/333370a0. [DOI] [PubMed] [Google Scholar]
  6. Bouvier M., Hnatowich M., Collins S., Kobilka B. K., Deblasi A., Lefkowitz R. J., Caron M. G. Expression of a human cDNA encoding the beta 2-adrenergic receptor in Chinese hamster fibroblasts (CHW): functionality and regulation of the expressed receptors. Mol Pharmacol. 1988 Feb;33(2):133–139. [PubMed] [Google Scholar]
  7. Chidiac P., Hebert T. E., Valiquette M., Dennis M., Bouvier M. Inverse agonist activity of beta-adrenergic antagonists. Mol Pharmacol. 1994 Mar;45(3):490–499. [PubMed] [Google Scholar]
  8. De Lean A., Stadel J. M., Lefkowitz R. J. A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled beta-adrenergic receptor. J Biol Chem. 1980 Aug 10;255(15):7108–7117. [PubMed] [Google Scholar]
  9. Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hausdorff W. P., Bouvier M., O'Dowd B. F., Irons G. P., Caron M. G., Lefkowitz R. J. Phosphorylation sites on two domains of the beta 2-adrenergic receptor are involved in distinct pathways of receptor desensitization. J Biol Chem. 1989 Jul 25;264(21):12657–12665. [PubMed] [Google Scholar]
  11. Hebert T. E., Moffett S., Morello J. P., Loisel T. P., Bichet D. G., Barret C., Bouvier M. A peptide derived from a beta2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation. J Biol Chem. 1996 Jul 5;271(27):16384–16392. doi: 10.1074/jbc.271.27.16384. [DOI] [PubMed] [Google Scholar]
  12. Heldin C. H. Dimerization of cell surface receptors in signal transduction. Cell. 1995 Jan 27;80(2):213–223. doi: 10.1016/0092-8674(95)90404-2. [DOI] [PubMed] [Google Scholar]
  13. Herberg J. T., Codina J., Rich K. A., Rojas F. J., Iyengar R. The hepatic glucagon receptor. Solubilization, characterization, and development of an affinity adsorption assay for the soluble receptor. J Biol Chem. 1984 Jul 25;259(14):9285–9294. [PubMed] [Google Scholar]
  14. Hirschberg B. T., Schimerlik M. I. A kinetic model for oxotremorine M binding to recombinant porcine m2 muscarinic receptors expressed in Chinese hamster ovary cells. J Biol Chem. 1994 Oct 21;269(42):26127–26135. [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Kobilka B. Adrenergic receptors as models for G protein-coupled receptors. Annu Rev Neurosci. 1992;15:87–114. doi: 10.1146/annurev.ne.15.030192.000511. [DOI] [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Limbird L. E., Meyts P. D., Lefkowitz R. J. Beta-adrenergic receptors: evidence for negative cooperativity. Biochem Biophys Res Commun. 1975 Jun 16;64(4):1160–1168. doi: 10.1016/0006-291x(75)90815-3. [DOI] [PubMed] [Google Scholar]
  20. Loisel T. P., Adam L., Hebert T. E., Bouvier M. Agonist stimulation increases the turnover rate of beta 2AR-bound palmitate and promotes receptor depalmitoylation. Biochemistry. 1996 Dec 10;35(49):15923–15932. doi: 10.1021/bi9611321. [DOI] [PubMed] [Google Scholar]
  21. Maggio R., Vogel Z., Wess J. Coexpression studies with mutant muscarinic/adrenergic receptors provide evidence for intermolecular "cross-talk" between G-protein-linked receptors. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):3103–3107. doi: 10.1073/pnas.90.7.3103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mattera R., Pitts B. J., Entman M. L., Birnbaumer L. Guanine nucleotide regulation of a mammalian myocardial muscarinic receptor system. Evidence for homo- and heterotropic cooperativity in ligand binding analyzed by computer-assisted curve fitting. J Biol Chem. 1985 Jun 25;260(12):7410–7421. [PubMed] [Google Scholar]
  23. 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]
  24. Moffett S., Adam L., Bonin H., Loisel T. P., Bouvier M., Mouillac B. Palmitoylated cysteine 341 modulates phosphorylation of the beta2-adrenergic receptor by the cAMP-dependent protein kinase. J Biol Chem. 1996 Aug 30;271(35):21490–21497. doi: 10.1074/jbc.271.35.21490. [DOI] [PubMed] [Google Scholar]
  25. Moffett S., Mouillac B., Bonin H., Bouvier M. Altered phosphorylation and desensitization patterns of a human beta 2-adrenergic receptor lacking the palmitoylated Cys341. EMBO J. 1993 Jan;12(1):349–356. doi: 10.1002/j.1460-2075.1993.tb05663.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Monnot C., Bihoreau C., Conchon S., Curnow K. M., Corvol P., Clauser E. Polar residues in the transmembrane domains of the type 1 angiotensin II receptor are required for binding and coupling. Reconstitution of the binding site by co-expression of two deficient mutants. J Biol Chem. 1996 Jan 19;271(3):1507–1513. doi: 10.1074/jbc.271.3.1507. [DOI] [PubMed] [Google Scholar]
  27. Mouillac B., Caron M., Bonin H., Dennis M., Bouvier M. Agonist-modulated palmitoylation of beta 2-adrenergic receptor in Sf9 cells. J Biol Chem. 1992 Oct 25;267(30):21733–21737. [PubMed] [Google Scholar]
  28. Neubig R. R. Membrane organization in G-protein mechanisms. FASEB J. 1994 Sep;8(12):939–946. doi: 10.1096/fasebj.8.12.8088459. [DOI] [PubMed] [Google Scholar]
  29. O'Dowd B. F., Hnatowich M., Caron M. G., Lefkowitz R. J., Bouvier M. Palmitoylation of the human beta 2-adrenergic receptor. Mutation of Cys341 in the carboxyl tail leads to an uncoupled nonpalmitoylated form of the receptor. J Biol Chem. 1989 May 5;264(13):7564–7569. [PubMed] [Google Scholar]
  30. Peterson G. L., Rosenbaum L. C., Broderick D. J., Schimerlik M. I. Physical properties of the purified cardiac muscarinic acetylcholine receptor. Biochemistry. 1986 Jun 3;25(11):3189–3202. doi: 10.1021/bi00359a017. [DOI] [PubMed] [Google Scholar]
  31. Potter L. T., Ballesteros L. A., Bichajian L. H., Ferrendelli C. A., Fisher A., Hanchett H. E., Zhang R. Evidence of paired M2 muscarinic receptors. Mol Pharmacol. 1991 Feb;39(2):211–221. [PubMed] [Google Scholar]
  32. Rodbell M. The role of GTP-binding proteins in signal transduction: from the sublimely simple to the conceptually complex. Curr Top Cell Regul. 1992;32:1–47. doi: 10.1016/b978-0-12-152832-4.50003-3. [DOI] [PubMed] [Google Scholar]
  33. Salomon Y., Londos C., Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem. 1974 Apr;58(2):541–548. doi: 10.1016/0003-2697(74)90222-x. [DOI] [PubMed] [Google Scholar]
  34. Shorr R. G., Lefkowitz R. J., Caron M. G. Purification of the beta-adrenergic receptor. Identification of the hormone binding subunit. J Biol Chem. 1981 Jun 10;256(11):5820–5826. [PubMed] [Google Scholar]
  35. Sinkins W. G., Kandel M., Kandel S. I., Schunack W., Wells J. W. G protein-linked receptors labeled by [3H]histamine in guinea pig cerebral cortex. I. Pharmacological characterization [corrected]. Mol Pharmacol. 1993 Apr;43(4):569–582. [PubMed] [Google Scholar]
  36. Sinkins W. G., Wells J. W. G protein-linked receptors labeled by [3H]histamine in guinea pig cerebral cortex. II. Mechanistic basis for multiple states of affinity [corrected]. Mol Pharmacol. 1993 Apr;43(4):583–594. [PubMed] [Google Scholar]
  37. Stoddard B. L., Biemann H. P., Koshland D. E., Jr Receptors and transmembrane signaling. Cold Spring Harb Symp Quant Biol. 1992;57:1–15. doi: 10.1101/sqb.1992.057.01.003. [DOI] [PubMed] [Google Scholar]
  38. Strosberg A. D. Structure, function, and regulation of adrenergic receptors. Protein Sci. 1993 Aug;2(8):1198–1209. doi: 10.1002/pro.5560020802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wessling-Resnick M., Johnson G. L. Allosteric behavior in transducin activation mediated by rhodopsin. Initial rate analysis of guanine nucleotide exchange. J Biol Chem. 1987 Mar 15;262(8):3697–3705. [PubMed] [Google Scholar]
  41. Willardson B. M., Pou B., Yoshida T., Bitensky M. W. Cooperative binding of the retinal rod G-protein, transducin, to light-activated rhodopsin. J Biol Chem. 1993 Mar 25;268(9):6371–6382. [PubMed] [Google Scholar]
  42. Wreggett K. A., Wells J. W. Cooperativity manifest in the binding properties of purified cardiac muscarinic receptors. J Biol Chem. 1995 Sep 22;270(38):22488–22499. doi: 10.1074/jbc.270.38.22488. [DOI] [PubMed] [Google Scholar]

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