Role of the cyclic AMP-dependent protein kinase in homologous resensitization of the beta1-adrenergic receptor.
A fundamental question in biology is how the various motifs in G protein-coupled receptors participate in the divergent functions orchestrated by these molecules. Here we describe a fundamental role for a serine residue at position 312 in the third intracellular loop of the human beta(1)-adrenergic receptor (beta(1)-AR) in endocytic recycling of the agonist-internalized receptor. In receptor recycling experiments that were monitored by confocal microscopy, the agonist-internalized wild-type (WT) beta(1)-AR recycled with a t(0.5) of 14 +/- 3 min. Mutagenesis of Ser(312) to alanine (Ser(312) --> Ala beta(1)-AR) or to the phosphoserine mimic aspartic acid (Ser(312) --> Asp beta(1)-AR) resulted in beta(1)-AR constructs that were pharmacologically indistinguishable from the WT beta(1)-AR. The internalized Ser(312) --> Asp beta(1)-AR recycled efficiently with a t(0.5) of 11 +/- 3 min, whereas the internalized Ser(312) --> Ala beta(1)-AR was not recycled or functionally resensitized through the endosomal pathway. Because this serine is a putative residue for phosphorylation by the cyclic AMP-dependent protein kinase (PKA), we examined the role of this kinase in recycling of the internalized beta(1)-AR. Inhibition of PKA biochemically or genetically using a dominant negative PKA construct blocked the recycling of the internalized WT beta(1)-AR. Phosphorylation studies revealed that the beta(1)-AR is partially phosphorylated by PKA and that phosphorylation of the beta(1)-AR by the catalytic subunit of PKA occurs exclusively at Ser(312). Our results identify a new signaling paradigm in which homologous activation of a kinase provides a reversible modification that shifts the itinerary of the internalized receptor toward recycling and resensitization. Therefore, PKA-mediated phosphorylation of G protein-coupled receptors might result in motif-dependent desensitization or resensitization.
Pharmacology and Molecular Sciences, University of Tennessee Health Sciences Center, 874 Union Avenue, Memphis, TN 38163, USA.
Lidia A
LA
Gardner
Noel M
NM
Delos Santos
Shannon G
SG
Matta
Michael A
MA
Whitt
Suleiman W
SW
Bahouth
D000262:Adenylyl Cyclases / Q000378:metabolism; D000322:Adrenergic Agonists / Q000494:pharmacology; D018674:Adrenergic Antagonists / Q000494:pharmacology; D000595:Amino Acid Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation; D018342:Receptors, Adrenergic, beta-1 / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D014162:Transfection
D000322:Adrenergic Agonists; D018674:Adrenergic Antagonists; D010455:Peptides; D018342:Receptors, Adrenergic, beta-1; D011994:Recombinant Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases; D007545:Isoproterenol