AKAP79-mediated targeting of the cyclic AMP-dependent protein kinase to the β1-adrenergic receptor promotes recycling and functional resensitization of the receptor

Abstract

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 β1-adrenergic receptor (β1-AR) were both necessary for efficient recycling and resensitization of the agonist-internalized β1-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 β1-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 β1-AR in HEK-293 cells and endogenously expressed β1-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 β1-AR in this cell line. Co-immunoprecipitations and fluorescence resonance energy transfer (FRET) microscopy experiments in HEK-293 cells revealed that the β1-AR, AKAP79, and PKA form a ternary complex at the carboxyl terminus of the β1-AR. This complex was involved in PKA-mediated phosphorylation of the third intracellular loop of the β1-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 β1-AR and thereby dictate the recycling and resensitization itineraries of the β1-AR. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.