Dynamic complexes of beta2-adrenergic receptors with protein kinases and phosphatases and the role of gravin.
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.
Shih
M
M
Department of Molecular Pharmacology, Diabetes & Metabolic Diseases Research Program, University Medical Center, State University of New York, Stony Brook, New York 11794-8651, USA.
Lin
F
F
Scott
J D
JD
Wang
H Y
HY
Malbon
C C
CC
D054758:A Kinase Anchor Proteins; D001324:Autoantigens / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D051552:beta-Adrenergic Receptor Kinases
D054758:A Kinase Anchor Proteins; C491162:AKAP12 protein, human; D001324:Autoantigens; D018797:Cell Cycle Proteins; D016376:Oligonucleotides, Antisense; D011506:Proteins; D018343:Receptors, Adrenergic, beta-2; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D051552:beta-Adrenergic Receptor Kinases; D010749:Phosphoprotein Phosphatases