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, 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 ...

Characterization of a Novel Ubiquitin-Conjugating Enzyme That Regulates β1,4-Galactosyltransferase-1 in Embryonic Stem Cells

“…Partial support for this hypothesis is that a small fraction of cell surface GalT1 has been cofractionated with caveolin in lipid rafts [21]. Furthermore, long GalT1 has been shown to associate with Src-suppressed C kinase substrate (SSeCKS), a previously defined kinase and cytoskeleton scaffolding protein important for agonist-induced internalization and resensitization of G-protein-linked receptors [20,35]. In this scenario, GalT1-associated GTAP may trigger ubiquitination and degradation of caveolin and/or SSeCKS, thereby affecting efficient internalization and function of GalT1.…”

AKAPs (A-kinase anchoring proteins) and molecules that compose their G-protein-coupled receptor signalling complexes

“…The AKAP domain provides a reversible, dynamic docking site for the β 2 AR and, presumably, other GPCRs. This would explain the basis for the association of AKAP250 as well as AKAP79 with β 2 AR [48,69]. We propose that, for AKAP79/250, this AKAP domain might be better considered as a 'GPCR-binding domain', reflecting its role in docking the β 2 AR and probably other GPCRs.…”

“…The binding of AKAP250 to β 2 AR is reversible, dynamically regulated by activation of the receptor (Figure 4) and essential for the normal subsequent sequellae in the desensitization, sequestration, resensitization and recycling of the β 2 AR [25,48]. For AKAP79, β 2 AR binding appears to be largely constitutive.…”

“…More detailed analysis of this observation [47] and the cellular context, however, will be needed to fully explore this topic. The suppression of AKAP250 expression by antisense technology leads to a disruption of β 2 AR trafficking and resensitization, demonstrating the central role of this scaffold in AKAP79/ 250-based β 2 AR signalling complexes [19,25,47,48]. In silico analysis of AKAP250 revealed three 'AKAP motifs' at residue positions 603-633, 752-782 and 797-827.…”

AKAP12/Gravin is inactivated by epigenetic mechanism in human gastric carcinoma and shows growth suppressor activity

“…AKAP12 organizes the complex of PKA and PKC (Nauert et al, 1997), and is an important regulator of the b 2 -adrenergic receptor complex (Shih et al, 1999;Lin et al, 2000a). AKAP12 expression can be induced by several drugs like phorbol ester (Gordon et al, 1992;Nauert et al, 1997) and lysophosphatidylcholine (Sato et al, 1998), which suggests the participation of AKAP12 in diverse signal transduction cascades.…”