Dynamic Complexes of β2-Adrenergic Receptors with Protein Kinases and Phosphatases and the Role of Gravin

Meiling Shih,Fubao Lin,John D Scott,Hsien-Yu Wang,Craig C Malbon

doi:10.1074/jbc.274.3.1588

Abstract

Signals mediated by G-protein-linked receptors display agonist-induced attenuation and recovery involving both protein kinases and phosphatases. The role of protein kinases and phosphatases in agonist-induced attenuation and recovery of beta-adrenergic receptors was explored by two complementary approaches, antisense RNA suppression and co-immunoprecipitation of target elements. Protein phosphatases 2A and 2B are associated with the unstimulated receptor, the latter displaying a transient decrease followed by a 2-fold increase in the levels of association at 30 min following challenge with agonist. Protein kinase A displays a robust, agonist-induced association with beta-adrenergic receptors over the same period. Suppression of phosphatases 2A and 2B with antisense RNA or inhibition of their activity with calyculin A and FK506, respectively, blocks resensitization following agonist removal. Recycling of receptors to the plasma membrane following agonist-promoted sequestration is severely impaired by loss of either phosphatase 2B or protein kinase C. In addition, loss of protein kinase C diminishes association of phosphatase 2B with beta-adrenergic receptors. Overlay assays performed with the RII subunit of protein kinase A and co-immunoprecipitations reveal proteins of the A kinase-anchoring proteins (AKAP) family, including AKAP250 also known as gravin, associated with the beta-adrenergic receptor. Suppression of gravin expression disrupts recovery from agonist-induced desensitization, confirming the role of gravin in organization of G-protein-linked signaling complexes. The Ht31 peptide, which blocks AKAP protein-protein interactions, blocks association of beta-adrenergic receptors with protein kinase A. These data are the first to reveal dynamic complexes of beta-adrenergic receptors with protein kinases and phosphatases acting via an anchoring protein, gravin.

Highlights

Agonist-induced desensitization involving at least three prominent kinase activities (2), cyclic AMP-dependent protein kinase, calcium and phospholipid-sensitive protein kinase, and members of the G-protein-linked receptor kinase family, like the ␤-adrenergic receptor kinase. ␤2-Adrenergic receptors (␤2AR) are substrates for protein kinase A, protein kinase C, G-protein-coupled receptor kinase, as well as growth factor receptors with intrinsic tyrosine kinase activity (3, 4)
Protein kinase C is shown to be obligate for resensitization of G-protein-linked receptors (GPLRs), its action blocked by protein kinase C deficiency and by the protein kinase C inhibitor bisindolylmaleimide, and mimicked by FK506, a protein phosphatase2B inhibitor
In the absence of exposure to the ␤-adrenergic agonist isoproterenol, immunoprecipitates of ␤-adrenergic receptors subjected to SDS-polyacrylamide gel electrophoresis and stained with antibodies to PP2B␣ reveal the presence of phosphatase PP2B in association with the ␤-adrenergic receptor (0-min desensitization time, Fig. 1A)

Summary

Introduction

Agonist-induced desensitization involving at least three prominent kinase activities (2), cyclic AMP-dependent protein kinase (protein kinase A), calcium and phospholipid-sensitive protein kinase (protein kinase C), and members of the G-protein-linked receptor kinase family, like the ␤-adrenergic receptor kinase. ␤2-Adrenergic receptors (␤2AR) are substrates for protein kinase A, protein kinase C, G-protein-coupled receptor kinase, as well as growth factor receptors with intrinsic tyrosine kinase activity (3, 4). Cells in which gravin was suppressed by antisense oligodeoxynucleotides were analyzed for Protein Kinase C and Receptor Recovery agonist-induced (isoproterenol) desensitization and resensitization following a wash-out of agonist (2).

Results

Conclusion