Biochemical characterization of phosphorylated beta-adrenergic receptors from catecholamine-desensitized turkey erythrocytes.
Isoproterenol-induced desensitization of turkey erythrocyte adenylate cyclase is accompanied (1) by a decrease in the mobility of beta-adrenergic receptor proteins, specifically photoaffinity labeled with 125I-(p-azidobenzyl)carazolol (125I-PABC), on sodium dodecyl sulfate (SDS)-polyacrylamide gels and (2) by a 2-3-fold increase in phosphate incorporation into the beta receptor [Stadel, J.M., Nambi, P., Shorr, R. G. L., Sawyer, D. F., Caron, M. G., & Lefkowitz, R. J. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3173]. Analysis of 32P-labeled beta receptors partially purified by affinity chromatography and subsequently hydrolyzed in 6 N HCl revealed that the beta receptor from control erythrocytes contained only phosphoserine and that agonist-promoted phosphorylation of the receptor in desensitized cells occurred on serine residues. Comparison of limited-digest peptide maps of 125I-PABC-labeled beta receptors from control and desensitized erythrocytes reveals distinctly different sensitivities of the two beta receptors to cleavage by chymotrypsin and Staphylococcus aureus protease. The altered mobility of the 125I-PABC-labeled beta receptor from desensitized erythrocytes was eliminated when 5 M urea was included in the SDS-polyacrylamide gels. Limited-digest peptide mapping of 32P-labeled beta receptors from control and desensitized cells with the protease papain identified a unique phosphorylated peptide in desensitized preparations. Our results are consistent with the hypothesis that the altered mobility of beta-receptor proteins on SDS gels following desensitization is due to changes in conformation promoted by prolonged exposure to agonists.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006868:Hydrolysis; D007457:Iodine Radioisotopes; D007545:Isoproterenol / Q000494:pharmacology*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D014422:Turkeys
D000596:Amino Acids; D007457:Iodine Radioisotopes; D010761:Phosphorus Radioisotopes; D011943:Receptors, Adrenergic, beta; D007545:Isoproterenol