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 scaf folds for a wide-range of protein-protein interactions. The 250-kDa AKAP kn own as gravin plays a central role in organizing G-protein-coupled receptor s to the protein kinases and phosphatases that regulate receptor function i n desensitization, resensitization, and sequestration. Although gravin is c ritical for G-protein-linked receptor biology, the molecular features of th e receptor necessary for interaction with this scaffold are not known. Here in, we map the regions of the beta (2)-adrenergic receptor that are require d for binding to gravin, Intracellular loops 1, 2, and 3 appear not to part icipate 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 r eadily for the binding of the beta (2)-adrenergic receptor by gravin, both using in vitro and in vivo assays. C-terminally truncated peptides with seq uences 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 Hi s-390 (61 residues), as well as N-terminally truncated peptides from Gln-39 1 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-rec eptor interaction was examined in response to agonist by confocal microscop y, Remarkably, the association of the receptor with gravin was not disrupte d during agonist-induced sequestration. The receptor-scaffold complex was m aintained during agonist-induced sequestration. These data, in agreement wi th the biochemical data, reveal that gravin binds the receptor through the beta2-adrenergic receptor C-terminal cytoplasmic domain and that this inter action is maintained as the receptor is internalized. This is the first rep ort of an AKAP scaffold protein translocating with its receptor, in this ca se a G-protein-coupled receptor.