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Desensitization of the turkey erythrocyte beta-adrenergic receptor in a cell-free system. Evidence that multiple protein kinases can phosphorylate and desensitize the receptor.

Publication ,  Journal Article
Nambi, P; Peters, JR; Sibley, DR; Lefkowitz, RJ
Published in: J Biol Chem
February 25, 1985

We have used a recently developed cell-free system (cell lysate) derived from turkey erythrocytes to explore the potential role of cAMP-activated and other protein kinase systems in desensitizing the adenylate cyclase-coupled beta-adrenergic receptor. Desensitization by the agonist isoproterenol required more than simple occupancy of the receptor by the agonist since under conditions where adenylate cyclase was not activated, no desensitization occurred. As in whole cells, addition of cyclic nucleotides to the cell lysate produced only approximately 50% of the maximal isoproterenol-induced desensitization obtainable. Addition of the purified cAMP-dependent protein kinase holoenzyme plus isoproterenol to isolated turkey erythrocyte plasma membranes mimicked the submaximal desensitization induced in lysates by cAMP. This effect was entirely blocked by the specific inhibitor of the cAMP-dependent protein kinase. By contrast, maximal desensitization induced in lysates by isoproterenol was only approximately 50% attenuated by the protein kinase inhibitor. In the lysate preparations, isoproterenol was also shown to induce, in a stereospecific fashion, phosphorylation of the beta-adrenergic receptor. Phosphorylation promoted by isoproterenol was attenuated by cAMP-dependent protein kinase inhibitor to the same extent as desensitization (i.e. approximately 50%). Phorbol diesters also promoted receptor desensitization and phosphorylation in cell lysates. The desensitization was mimicked by incubation of isolated turkey erythrocyte membranes with partially purified preparations of protein kinase C plus phorbol diesters. In the cell lysate, calmodulin also promoted receptor phosphorylation and desensitization which was blocked by EGTA. Desensitization of adenylate cyclase by isoproterenol, phorbol diesters, and calmodulin was not observed to be additive. These findings suggest that: (a) multiple protein kinase systems, including cAMP-dependent, protein kinase C-dependent, and Ca2+/calmodulin-dependent kinases, are capable of regulating beta-adrenergic receptor function via phosphorylation reactions and that (b) cAMP may not be the sole mediator of isoproterenol-induced phosphorylation and desensitization in these cells.

Duke Scholars

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

February 25, 1985

Volume

260

Issue

4

Start / End Page

2165 / 2171

Location

United States

Related Subject Headings

  • Turkeys
  • Receptors, Adrenergic, beta
  • Protein Kinases
  • Protein Kinase C
  • Phosphorylation
  • Phorbol Esters
  • Isoproterenol
  • Erythrocytes
  • Cyclic AMP
  • Cell-Free System
 

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

February 25, 1985

Volume

260

Issue

4

Start / End Page

2165 / 2171

Location

United States

Related Subject Headings

  • Turkeys
  • Receptors, Adrenergic, beta
  • Protein Kinases
  • Protein Kinase C
  • Phosphorylation
  • Phorbol Esters
  • Isoproterenol
  • Erythrocytes
  • Cyclic AMP
  • Cell-Free System