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
. 1983 May;80(10):2849–2853. doi: 10.1073/pnas.80.10.2849

Photoaffinity labeling of the beta-adrenergic receptor from cultured lymphoma cells with [125I]iodoazidobenzylpindolol: loss of the label with desensitization.

A Rashidbaigi, A E Ruoho, D A Green, R B Clark
PMCID: PMC393929  PMID: 6134281

Abstract

The beta-adrenergic antagonist [125I]iodoazidobenzylpindolol ( [125I]IABP) specifically photolabeled two polypeptides in membrane preparations from wild-type (WT) and coupling protein-deficient cyc- cultured lymphoma cells. The molecular weights of the two polypeptides determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis were 65,000 and 55,000. They were labeled in a ratio of approximately 1:1. Pretreatment of intact WT or cyc- cells with 1.0 microM epinephrine for 15 min (desensitization) resulted in a greater loss of the 55,000 Mr polypeptide (40-60%) relative to the 65,000 Mr peptide (10-30% loss). An 18- to 24-hr pretreatment of WT cells with terbutaline (down-regulation) led to a greater than 90% reduction of the photolabeling of both polypeptides, whereas a similar pretreatment of cyc- cells resulted in no further loss of labeled receptor than that observed after only a 15-min pretreatment with epinephrine. There was no indication of a change in the electrophoretic mobility of the [125I]IABP-labeled receptors after either short- or long-term agonist pretreatment. These data provide direct evidence for heterogeneity of the beta-adrenergic receptor in lymphoma cells. The differential loss of the [125I]IABP labeling in the two polypeptides suggests a functional heterogeneity as well.

Full text

PDF
2849

Images in this article

2851Image
on p.2851

Selected References

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

  1. Brunton L. L., Maguire M. E., Anderson H. J., Gilman A. G. Expression of genes for metabolism of cyclic adenosine 3':5'-monophosphate in somatic cells. beta-Adrenergic and PGE1 receptors in parental and hybrid cells. J Biol Chem. 1977 Feb 25;252(4):1293–1302. [PubMed] [Google Scholar]
  2. Chowdhry V., Westheimer F. H. Photoaffinity labeling of biological systems. Annu Rev Biochem. 1979;48:293–325. doi: 10.1146/annurev.bi.48.070179.001453. [DOI] [PubMed] [Google Scholar]
  3. Clark R. B., Goka T. J., Green D. A., Barber R., Butcher R. W. Differences in the forskolin activation of adenylate cyclases in wild-type and variant lymphoma cells. Mol Pharmacol. 1982 Nov;22(3):609–613. [PubMed] [Google Scholar]
  4. Dibner M. D., Insel P. A. Serum catecholamines desensitize beta-adrenergic receptors of cultured C6 glioma cells. J Biol Chem. 1981 Jul 25;256(14):7343–7346. [PubMed] [Google Scholar]
  5. Green D. A., Clark R. B. Adenylate cyclase coupling proteins are not essential for agonist-specific desensitization of lymphoma cells. J Biol Chem. 1981 Mar 10;256(5):2105–2108. [PubMed] [Google Scholar]
  6. Green D. A., Friedman J., Clark R. B. Epinephrine desensitization of adenylate cyclase from cyc- and S49 cultured lymphoma cells. J Cyclic Nucleotide Res. 1981;7(3):161–172. [PubMed] [Google Scholar]
  7. Harden T. K., Cotton C. U., Waldo G. L., Lutton J. K., Perkins J. P. Catecholamine-induced alteration in sedimentation behavior of membrane bound beta-adrenergic receptors. Science. 1980 Oct;210(4468):441–443. doi: 10.1126/science.6254143. [DOI] [PubMed] [Google Scholar]
  8. Iyengar R., Bhat M. K., Riser M. E., Birnbaumer L. Receptor-specific desensitization of the S49 lymphoma cell adenylyl cyclase. Unaltered behavior of the regulatory component. J Biol Chem. 1981 May 25;256(10):4810–4815. [PubMed] [Google Scholar]
  9. Johnson G. L., Kaslow H. R., Bourne H. R. Genetic evidence that cholera toxin substrates are regulatory components of adenylate cyclase. J Biol Chem. 1978 Oct 25;253(20):7120–7123. [PubMed] [Google Scholar]
  10. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  11. 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]
  12. Lambin P., Rochu D., Fine J. M. A new method for determination of molecular weights of proteins by electrophoresis across a sodium dodecyl sulfate (SDS)-polyacrylamide gradient gel. Anal Biochem. 1976 Aug;74(2):567–575. doi: 10.1016/0003-2697(76)90239-6. [DOI] [PubMed] [Google Scholar]
  13. Rashidbaigi A., Ruoho A. E. Iodoazidobenzylpindolol, a photoaffinity probe for the beta-adrenergic receptor. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1609–1613. doi: 10.1073/pnas.78.3.1609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Rashidbaigi A., Ruoho A. E. Photoaffinity labeling of beta-adrenergic receptors: identification of the beta-receptor binding site(s) from turkey, pigeon, and frog erythrocyte. Biochem Biophys Res Commun. 1982 May 14;106(1):139–148. doi: 10.1016/0006-291x(82)92069-1. [DOI] [PubMed] [Google Scholar]
  15. Rashidbaigi A., Ruoho A. E. Synthesis and characterization of iodoazidobenzylpindolol. J Pharm Sci. 1982 Mar;71(3):305–307. doi: 10.1002/jps.2600710310. [DOI] [PubMed] [Google Scholar]
  16. Ross E. M., Gilman A. G. Biochemical properties of hormone-sensitive adenylate cyclase. Annu Rev Biochem. 1980;49:533–564. doi: 10.1146/annurev.bi.49.070180.002533. [DOI] [PubMed] [Google Scholar]
  17. Ruoho A. E., Kiefer H., Roeder P. E., Singer S. J. The mechanism of photoaffinity labeling. Proc Natl Acad Sci U S A. 1973 Sep;70(9):2567–2571. doi: 10.1073/pnas.70.9.2567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Shear M., Insel P. A., Melmon K. L., Coffino P. Agonist-specific refractoriness induced by isoproterenol. Studies with mutant cells. J Biol Chem. 1976 Dec 10;251(23):7572–7576. [PubMed] [Google Scholar]
  19. Stadel J. M., De Lean A., Mullikin-Kilpatrick D., Sawyer D. D., Lefkowitz R. J. Catecholamine-induced desensitization in turkey erythrocytes: cAMP mediated impairment of high affinity agonist binding without alteration in receptor number. J Cyclic Nucleotide Res. 1981;7(1):37–47. [PubMed] [Google Scholar]
  20. Stadel J. M., Nambi P., Lavin T. N., Heald S. L., Caron M. G., Lefkowitz R. J. Catecholamine-induced desensitization of turkey erythrocyte adenylate cyclase. Structural alterations in the beta-adrenergic receptor revealed by photoaffinity labeling. J Biol Chem. 1982 Aug 25;257(16):9242–9245. [PubMed] [Google Scholar]
  21. Sternweis P. C., Gilman A. G. Reconstitution of catecholamine-sensitive adenylate cyclase. Reconstitution of the uncoupled variant of the S40 lymphoma cell. J Biol Chem. 1979 May 10;254(9):3333–3340. [PubMed] [Google Scholar]
  22. Su Y. F., Harden T. K., Perkins J. P. Catecholamine-specific desensitization of adenylate cyclase. Evidence for a multistep process. J Biol Chem. 1980 Aug 10;255(15):7410–7419. [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