The scaffold protein gravin (cAMP-dependent protein kinase-anchoring protein 250) binds the beta 2-adrenergic receptor via the receptor cytoplasmic Arg-329 to Leu-413 domain and provides a mobile scaffold during desensitization.
The cyclic AMP-dependent kinase-anchoring proteins (AKAPs) function as scaffolds for a wide-range of protein-protein interactions. The 250-kDa AKAP known as gravin plays a central role in organizing G-protein-coupled receptors to the protein kinases and phosphatases that regulate receptor function in desensitization, resensitization, and sequestration. Although gravin is critical for G-protein-linked receptor biology, the molecular features of the receptor necessary for interaction with this scaffold are not known. Herein, we map the regions of the beta(2)-adrenergic receptor that are required for binding to gravin. Intracellular loops 1, 2, and 3 appear not to participate in the binding of the receptor to the scaffold. In contrast, the C-terminal cytoplasmic region of the receptor (Arg-329 to Leu-413) competes readily for the binding of the beta(2)-adrenergic receptor by gravin, both using in vitro and in vivo assays. C-terminally truncated peptides with sequences ranging from Arg-329 to Leu-342 (13 aminoacyl residues), to Asn-352 (23 residues), to Tyr-366 (37 residues), to Asp-380 (51 residues), or to His-390 (61 residues), as well as N-terminally truncated peptides from Gln-391 to Leu-413 (23 residues) or Leu-381 to Leu-413 (33 residues) displayed no ability to block binding of receptor to gravin. The combination of Arg-329 to His-390 peptide and Gln-391 to Leu-413 peptide, however, reconstitutes a fragmented but full-length C-terminal region and also potently blocks the ability of gravin to bind the beta(2)-adrenergic receptor. The gravin-receptor interaction was examined in response to agonist by confocal microscopy. Remarkably, the association of the receptor with gravin was not disrupted during agonist-induced sequestration. The receptor-scaffold complex was maintained during agonist-induced sequestration. These data, in agreement with the biochemical data, reveal that gravin binds the receptor through the beta(2)-adrenergic receptor C-terminal cytoplasmic domain and that this interaction is maintained as the receptor is internalized. This is the first report of an AKAP scaffold protein translocating with its receptor, in this case a G-protein-coupled receptor.
Fan
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Department of Molecular Pharmacology, Diabetes & Metabolic Diseases Research Program, University Medical Center, SUNY/Stony Brook, Stony Brook, New York 11794-8651, USA.
Shumay
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Wang
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Malbon
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D054758:A Kinase Anchor Proteins; D001120:Arginine / Q000737:chemistry; D019390:Arrestins / Q000378:metabolism; D001665:Binding Sites; D018797:Cell Cycle Proteins; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D015536:Down-Regulation; D054769:G-Protein-Coupled Receptor Kinase 2; D006801:Humans; D007930:Leucine / Q000737:chemistry; D046911:Macromolecular Substances; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010446:Peptide Fragments / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D051552:beta-Adrenergic Receptor Kinases; D000071557:beta-Arrestins
D054758:A Kinase Anchor Proteins; C491162:AKAP12 protein, human; D019390:Arrestins; D018797:Cell Cycle Proteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011506:Proteins; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; D000071557:beta-Arrestins; D001120:Arginine; D017868:Cyclic AMP-Dependent Protein Kinases; C496567:GRK2 protein, human; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2; D007930:Leucine