Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 281, Issue 44, Pages 33537-33553Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M601809200
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Funding
- NHLBI NIH HHS [HL-71419] Funding Source: Medline
- NIGMS NIH HHS [GM-18231] Funding Source: Medline
- NINDS NIH HHS [NS-46661] Funding Source: Medline
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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.
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