Gravin-mediated formation of signaling complexes in β2-adrenergic receptor desensitization and resensitization

Abstract

Agonist-induced desensitization and resensitization of G-protein-linked receptors involve the interaction of receptors with protein kinases, phosphatase, β-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 β-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, β-arrestin, and clathrin. Confocal microscopy of the green fluorescence protein-tagged β2- 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, β-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/phosphatase, β- arrestin, and clathrin is essential in agonist-induced internalization and resensitization of G-protein-linked receptors.