AKAP79-mediated targeting of the cyclic AMP-dependent protein kinase to the beta1-adrenergic receptor promotes recycling and functional resensitization of the receptor.
Resensitization of G protein-coupled receptors (GPCR) following prolonged agonist exposure is critical for restoring the responsiveness of the receptor to subsequent challenges by agonist. The 3'-5' cyclic AMP-dependent protein kinase (PKA) and serine 312 in the third intracellular loop of the human beta(1)-adrenergic receptor (beta(1)-AR) were both necessary for efficient recycling and resensitization of the agonist-internalized beta(1)-AR (Gardner, L. A., Delos Santos, N. M., Matta, S. G., Whitt, M. A., and Bahouth, S. W. (2004) J. Biol. Chem. 279, 21135-21143). Because PKA is compartmentalized near target substrates by interacting with protein kinase A anchoring proteins (AKAPs), the present study was undertaken to identify the AKAP involved in PKA-mediated phosphorylation of the beta(1)-AR and in its recycling and resensitization. Here, we report that Ht-31 peptide-mediated disruption of PKA/AKAP interactions prevented the recycling and functional resensitization of heterologously expressed beta(1)-AR in HEK-293 cells and endogenously expressed beta(1)-AR in SK-N-MC cells and neonatal rat cortical neurons. Whereas several endogenous AKAPs were identified in HEK-293 cells, small interfering RNA-mediated down-regulation of AKAP79 prevented the recycling of the beta(1)-AR in this cell line. Co-immunoprecipitations and fluorescence resonance energy transfer (FRET) microscopy experiments in HEK-293 cells revealed that the beta(1)-AR, AKAP79, and PKA form a ternary complex at the carboxyl terminus of the beta(1)-AR. This complex was involved in PKA-mediated phosphorylation of the third intracellular loop of the beta(1)-AR because disruption of PKA/AKAP interactions or small interfering RNA-mediated down-regulation of AKAP79 both inhibited this response. Thus, AKAP79 provides PKA to phosphorylate the beta(1)-AR and thereby dictate the recycling and resensitization itineraries of the beta(1)-AR.
Gardner
Lidia A
LA
Department of Pharmacology, University of Tennessee Health Sciences Center, Memphis, Tennessee 38163, USA.
Tavalin
Steven J
SJ
Goehring
April S
AS
Scott
John D
JD
Bahouth
Suleiman W
SW
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D058665:Adrenergic beta-1 Receptor Agonists; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering; D018342:Receptors, Adrenergic, beta-1 / Q000235:genetics / Q000378:metabolism*
D054758:A Kinase Anchor Proteins; C487775:AKAP5 protein, human; D048868:Adaptor Proteins, Signal Transducing; D058665:Adrenergic beta-1 Receptor Agonists; D021122:Protein Subunits; D034741:RNA, Small Interfering; D018342:Receptors, Adrenergic, beta-1; D017868:Cyclic AMP-Dependent Protein Kinases