Agonist-induced desensitization and resensitization of G-protein-linked receptors involve the interaction of receptors with protein kinases, phosphatases, -arrestin, and clathrin organized by at least one scaffold protein. The dynamic composition of the signaling complexes and the role of the scaffold protein AKAP250 (gravin) in agonist-induced attenuation and recovery of -adrenergic receptors were explored by co-immunoprecipitation of target elements, antisense suppression, and confocal microscopy. Gravin associated with unstimulated receptor, and the association was increased significantly after agonist stimulation for up to 60 min. Agonist stimulation also induced a robust association of the receptor-gravin complex with protein kinases A and C, G-protein-linked receptor kinase-2, -arrestin, and clathrin. Confocal microscopy of the green fluorescence protein-tagged  2 -adrenergic receptor showed that the receptor underwent sequestration after agonist stimulation. Suppression of gravin expression via antisense oligodeoxynucleotides disrupted agonist-induced association of the receptor with G-protein-linked receptor kinase-2, -arrestin, and clathrin as well as receptor recovery from desensitization. Gravin deficiency also inhibited agonist-induced sequestration. These data reveal that gravin-mediated formation of signaling complexes with protein kinases/phosphatases, -arrestin, and clathrin is essential in agonist-induced internalization and resensitization of G-protein-linked receptors.
G-protein-linked receptors (GPLRs)1 display attenuation of the receptor-mediated signal or "desensitization" and then "resensitization" after removal of agonist (1, 2). Protein phosphorylation is a critical element of agonist-induced desensitization involving at least three prominent kinase activities: cyclic AMPdependent protein kinase (protein kinase A), calcium-and phospholipid-sensitive protein kinase (protein kinase C), and members of the G-protein-linked receptor kinase family (GRK) such as the -adrenergic receptor kinases (2).  2 -Adrenergic receptors are substrates for protein kinases A and C and Gprotein-linked receptor kinases as well as growth factor receptors with intrinsic tyrosine kinase activity (3, 4). Recent studies revealed that some G-protein-linked receptors such as the  2 -adrenergic receptor, m1 muscarinic cholinergic receptor, luteinizing hormone/human chorionic gonadotropin receptor, and gastrin-releasing peptide receptor undergo sequestration via clathrin-dependent endocytosis after agonist stimulation (5-7). -Arrestin has been found to play an important role in targeting the receptors to clathrin-coated pits (8 -11). Following phosphorylation of the agonist-occupied receptors by GRKs, -arrestin binds to the receptors, thereby terminating signaling transduction via endocytosis (10).Agonist-induced desensitization and sequestration of GPLRs are a complex process involving phosphorylation of the receptor by various protein kinases, followed by the interaction of the receptor with other proteins ...