Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Apr 15;90(8):3665–3669. doi: 10.1073/pnas.90.8.3665

Structural basis for receptor subtype-specific regulation revealed by a chimeric beta 3/beta 2-adrenergic receptor.

S B Liggett 1, N J Freedman 1, D A Schwinn 1, R J Lefkowitz 1
PMCID: PMC46362  PMID: 8386380

Abstract

The physiological significance of multiple G-protein-coupled receptor subtypes, such as the beta-adrenergic receptors (beta ARs), remains obscure, since in many cases several subtypes activate the same effector and utilize the same physiological agonists. We inspected the deduced amino acid sequences of the beta AR subtypes for variations in the determinants for agonist regulation as a potential basis for subtype differentiation. Whereas the beta 2AR has a C terminus containing 11 serine and threonine residues representing potential sites for beta AR kinase phosphorylation, which mediates rapid agonist-promoted desensitization, only 3 serines are present in the comparable region of the beta 3AR, and they are in a nonfavorable context. The beta 3AR also lacks sequence homology in regions which are important for agonist-mediated sequestration and down-regulation of the beta 2AR, although such determinants are less well defined. We therefore tested the idea that the agonist-induced regulatory properties of the two receptors might differ by expressing both subtypes in CHW cells and exposing them to the agonist isoproterenol. The beta 3AR did not display short-term agonist-promoted functional desensitization or sequestration, or long-term down-regulation. To assign a structural basis for these subtype-specific differences in agonist regulation, we constructed a chimeric beta 3/beta 2AR which comprised the beta 3AR up to proline-365 of the cytoplasmic tail and the C terminus of the beta 2AR. When cells expressing this chimeric beta 3/beta 2AR were exposed to isoproterenol, functional desensitization was observed. Whole-cell phosphorylation studies showed that the beta 2AR displayed agonist-dependent phosphorylation, but no such phosphorylation could be demonstrated with the beta 3AR, even when beta AR kinase was overexpressed. In contrast, the chimeric beta 3/beta 2AR did display agonist-dependent phosphorylation, consistent with its functional desensitization. In addition to conferring functional desensitization and phosphorylation to the beta 3AR, the C-terminal tail of the beta 2AR also conferred agonist-promoted sequestration and long-term receptor down-regulation.

Full text

PDF
3665

Images in this article

3667Fig. 3
on p.3667

Selected References

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

  1. Benovic J. L., DeBlasi A., Stone W. C., Caron M. G., Lefkowitz R. J. Beta-adrenergic receptor kinase: primary structure delineates a multigene family. Science. 1989 Oct 13;246(4927):235–240. doi: 10.1126/science.2552582. [DOI] [PubMed] [Google Scholar]
  2. Bouvier M., Collins S., O'Dowd B. F., Campbell P. T., de Blasi A., Kobilka B. K., MacGregor C., Irons G. P., Caron M. G., Lefkowitz R. J. Two distinct pathways for cAMP-mediated down-regulation of the beta 2-adrenergic receptor. Phosphorylation of the receptor and regulation of its mRNA level. J Biol Chem. 1989 Oct 5;264(28):16786–16792. [PubMed] [Google Scholar]
  3. 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]
  4. Campbell P. T., Hnatowich M., O'Dowd B. F., Caron M. G., Lefkowitz R. J., Hausdorff W. P. Mutations of the human beta 2-adrenergic receptor that impair coupling to Gs interfere with receptor down-regulation but not sequestration. Mol Pharmacol. 1991 Feb;39(2):192–198. [PubMed] [Google Scholar]
  5. Clark R. B., Friedman J., Dixon R. A., Strader C. D. Identification of a specific site required for rapid heterologous desensitization of the beta-adrenergic receptor by cAMP-dependent protein kinase. Mol Pharmacol. 1989 Sep;36(3):343–348. [PubMed] [Google Scholar]
  6. Cullen B. R. Use of eukaryotic expression technology in the functional analysis of cloned genes. Methods Enzymol. 1987;152:684–704. doi: 10.1016/0076-6879(87)52074-2. [DOI] [PubMed] [Google Scholar]
  7. Emorine L. J., Marullo S., Briend-Sutren M. M., Patey G., Tate K., Delavier-Klutchko C., Strosberg A. D. Molecular characterization of the human beta 3-adrenergic receptor. Science. 1989 Sep 8;245(4922):1118–1121. doi: 10.1126/science.2570461. [DOI] [PubMed] [Google Scholar]
  8. Frielle T., Collins S., Daniel K. W., Caron M. G., Lefkowitz R. J., Kobilka B. K. Cloning of the cDNA for the human beta 1-adrenergic receptor. Proc Natl Acad Sci U S A. 1987 Nov;84(22):7920–7924. doi: 10.1073/pnas.84.22.7920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Frielle T., Daniel K. W., Caron M. G., Lefkowitz R. J. Structural basis of beta-adrenergic receptor subtype specificity studied with chimeric beta 1/beta 2-adrenergic receptors. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9494–9498. doi: 10.1073/pnas.85.24.9494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Granneman J. G. Effects of agonist exposure on the coupling of beta 1 and beta 3 adrenergic receptors to adenylyl cyclase in isolated adipocytes. J Pharmacol Exp Ther. 1992 May;261(2):638–642. [PubMed] [Google Scholar]
  11. Granneman J. G., Lahners K. N. Differential adrenergic regulation of beta 1- and beta 3-adrenoreceptor messenger ribonucleic acids in adipose tissues. Endocrinology. 1992 Jan;130(1):109–114. doi: 10.1210/endo.130.1.1309320. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Hausdorff W. P., Campbell P. T., Ostrowski J., Yu S. S., Caron M. G., Lefkowitz R. J. A small region of the beta-adrenergic receptor is selectively involved in its rapid regulation. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):2979–2983. doi: 10.1073/pnas.88.8.2979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kobilka B. K., Dixon R. A., Frielle T., Dohlman H. G., Bolanowski M. A., Sigal I. S., Yang-Feng T. L., Francke U., Caron M. G., Lefkowitz R. J. cDNA for the human beta 2-adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor. Proc Natl Acad Sci U S A. 1987 Jan;84(1):46–50. doi: 10.1073/pnas.84.1.46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kobilka B. K., Kobilka T. S., Daniel K., Regan J. W., Caron M. G., Lefkowitz R. J. Chimeric alpha 2-,beta 2-adrenergic receptors: delineation of domains involved in effector coupling and ligand binding specificity. Science. 1988 Jun 3;240(4857):1310–1316. doi: 10.1126/science.2836950. [DOI] [PubMed] [Google Scholar]
  16. Liggett S. B., Bouvier M., Hausdorff W. P., O'Dowd B., Caron M. G., Lefkowitz R. J. Altered patterns of agonist-stimulated cAMP accumulation in cells expressing mutant beta 2-adrenergic receptors lacking phosphorylation sites. Mol Pharmacol. 1989 Oct;36(4):641–646. [PubMed] [Google Scholar]
  17. Liggett S. B. Functional properties of the rat and human beta 3-adrenergic receptors: differential agonist activation of recombinant receptors in Chinese hamster ovary cells. Mol Pharmacol. 1992 Oct;42(4):634–637. [PubMed] [Google Scholar]
  18. Liggett S. B., Ostrowski J., Chesnut L. C., Kurose H., Raymond J. R., Caron M. G., Lefkowitz R. J. Sites in the third intracellular loop of the alpha 2A-adrenergic receptor confer short term agonist-promoted desensitization. Evidence for a receptor kinase-mediated mechanism. J Biol Chem. 1992 Mar 5;267(7):4740–4746. [PubMed] [Google Scholar]
  19. Lohse M. J., Benovic J. L., Caron M. G., Lefkowitz R. J. Multiple pathways of rapid beta 2-adrenergic receptor desensitization. Delineation with specific inhibitors. J Biol Chem. 1990 Feb 25;265(6):3202–3211. [PubMed] [Google Scholar]
  20. Niman H. L., Houghten R. A., Walker L. E., Reisfeld R. A., Wilson I. A., Hogle J. M., Lerner R. A. Generation of protein-reactive antibodies by short peptides is an event of high frequency: implications for the structural basis of immune recognition. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4949–4953. doi: 10.1073/pnas.80.16.4949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Onorato J. J., Palczewski K., Regan J. W., Caron M. G., Lefkowitz R. J., Benovic J. L. Role of acidic amino acids in peptide substrates of the beta-adrenergic receptor kinase and rhodopsin kinase. Biochemistry. 1991 May 28;30(21):5118–5125. doi: 10.1021/bi00235a002. [DOI] [PubMed] [Google Scholar]
  22. Thomas R. F., Holt B. D., Schwinn D. A., Liggett S. B. Long-term agonist exposure induces upregulation of beta 3-adrenergic receptor expression via multiple cAMP response elements. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4490–4494. doi: 10.1073/pnas.89.10.4490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Valiquette M., Bonin H., Hnatowich M., Caron M. G., Lefkowitz R. J., Bouvier M. Involvement of tyrosine residues located in the carboxyl tail of the human beta 2-adrenergic receptor in agonist-induced down-regulation of the receptor. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5089–5093. doi: 10.1073/pnas.87.13.5089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Yu S. S., Lefkowitz R. J., Hausdorff W. P. Beta-adrenergic receptor sequestration. A potential mechanism of receptor resensitization. J Biol Chem. 1993 Jan 5;268(1):337–341. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES