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 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.
Highlights
The 1-AR2 is a major receptor for the physiological regulation of cardiac function by the sympathetic nervous system and plays an important role in clinical management of hypertension and heart failure (2)
protein kinase (PKA) Anchoring Is Essential for Recycling and Resensitization of the Human 1-AR—To test whether PKA/AKAP interactions are necessary for PKA-mediated recycling of the agonistinternalized 1-AR, the association between PKA and AKAP was perturbed with the st-Ht31 peptide. st-Ht31 is a cell-permeable peptide that contains the critical RII-binding domain common to all AKAPs and globally disrupts PKA-AKAP interactions, whereas the st-Ht31-proline does not disrupt their association and is used as a control (31, 35)
Prolonged or repeated activation of many G protein-coupled receptors (GPCR) induces rapid desensitization followed by a period during which the receptor is either resensitized or degraded (4, 7, 12)
Summary
Cell Cultures—Human embryonic kidney 293 (HEK-293) cells and neuroepithelioma SK-N-MC cells were obtained from American Type Culture Collection (Manassas, VA). Acid Strip Confocal Recycling Microscopy Protocol— HEK-293 cells stably expressing the FLAG- or Myc-tagged WT 1-AR were grown on poly-L-lysine-coated glass coverslips and serum-starved at 37 °C for 1 h in DMEM supplemented with 25 mM HEPES, pH 7.4. Cells for desensitization were exposed to 1 mM ascorbic acid (control) or 10 M isoproterenol for 10 min at 37 °C, and washed with serum-free DMEM supplemented with 10 mM. To determine the effect of disrupting AKAP-PKA interactions on isoproterenol-mediated phosphorylation of the 1-AR, HEK-293 cells expressing the WT 1-AR were pretreated with 50 M st-Ht31 or 50 M st-Ht31-pro for 30 min. To determine the effect of down-regulating AKAP79 on isoproterenol-mediated phosphorylation of the 1-AR, HEK-293 cells expressing the WT 1-AR were transiently transfected with 100 nM control or AKAP79 siRNA for 2 days. At the end of the run the gel was electroblotted to nitrocellulose and the filters were counted by the Instantimager, exposed to an x-ray film overnight (34)
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