The ability of a system to regulate its responsiveness in the presence of a continuous stimulus, often termed desensitization, has been extensively characterized for the beta2-adrenergic receptor (beta2AR). beta2AR signalling is rapidly attenuated through receptor phosphorylation and subsequent binding of the protein beta-arrestin. Ultimately the receptor undergoes internalization, and although the molecular mechanism is unclear, receptor phosphorylation and beta-arrestin binding have been implicated in this processs. Here we report that beta-arrestin and arrestin-3, but not visual arrestin, promote beta2AR internalization and bind with high affinity directly and stoichiometrically to clathrin, the major structural protein of coated pits. Moreover, beta-arrestin/arrestin chimaeras that are defective in either beta2AR or clathrin binding show a reduced ability to promote beta2AR endocytosis. Immunofluorescence microscopy of intact cells indicates an agonist-dependent colocalization of the beta2AR and beta-arrestin with clathrin. These results show that beta-arrestin functions as an adaptor in the receptor-mediated endocytosis pathway, and suggest a general mechanism for regulating the trafficking of G-protein-coupled receptors.
G protein-coupled receptors (GPRs) play a key role in controlling hormonal regulation of numerous second-messenger pathways. However, following agonist activation, most GPRs rapidly lose their ability to respond to hormone. For many GPRs, this process, commonly referred to as desensitization, appears to be primarily mediated by two protein families: G protein-coupled receptor kinases (GRKs) and arrestins. GRKs specifically bind to the agonist-occupied receptor, thereby promoting receptor phosphorylation, which in turn leads to arrestin binding. Arrestin binding precludes receptor/G protein interaction leading to functional desensitization. Many GPRs are then removed from the plasma membrane via clathrin-mediated endocytosis. Recent studies have implicated endocytosis in the resensitization of GPRs and have linked both GRKs and arrestins to this process. In this review, we discuss the role of GRKs and arrestins in regulating agonist-specific signaling and trafficking of GPRs.
Internalization of agonist-activated G protein-coupled receptors is mediated by non-visual arrestins, which also bind to clathrin and are therefore thought to act as adaptors in the endocytosis process. Phosphoinositides have been implicated in the regulation of intracellular receptor trafficking, and are known to bind to other coat components including AP-2, AP180 and COPI coatomer. Given these observations, we explored the possibility that phosphoinositides play a role in arrestin's function as an adaptor. High-affinity binding sites for phosphoinositides in β-arrestin (arrestin2) and arrestin3 (β-arrestin2) were identified, and dissimilar effects of phosphoinositide and inositol phosphate on arrestin interactions with clathrin and receptor were characterized. Alteration of three basic residues in arrestin3 abolished phosphoinositide binding with complete retention of clathrin and receptor binding. Unlike native protein, upon agonist activation, this mutant arrestin3 expressed in COS1 cells neither supported β 2 -adrenergic receptor internalization nor did it concentrate in coated pits, although it was recruited to the plasma membrane. These findings indicate that phosphoinositide binding plays a critical regulatory role in delivery of the receptor-arrestin complex to coated pits, perhaps by providing, with activated receptor, a multi-point attachment of arrestin to the plasma membrane.
The intracellular domains of G-protein-coupled receptors provide sites for interaction with key proteins involved in signal initiation and termination. As an initial approach to identify proteins interacting with these receptors and the receptor motifs required for such interactions, we used intracellular subdomains of G-protein- G-protein-coupled receptors possess a characteristic seven segments of hydrophobic amino acids that likely serve as membrane spans to form a core motif important for ligand recognition. The interaction of agonist with the receptor initiates an ill-defined conformational adjustment in this core motif, which is propagated to intracellular domains of the receptor resulting in the activation of G-protein and the initiation of intracellular signaling events. For most members of the superfamily of Gprotein-coupled receptors, the third intracellular (i3) 1 loop and the carboxyl-terminal tail of the receptor are key sites for signal initiation and termination, and these receptor domains also exhibit the greatest variability in size among different subfamilies of these receptors. The largest i3 domains (100 -240 amino acids) are found in receptors coupled to the G i , G o , and/or G q family of G-proteins (i.e. muscarinic, ␣-adrenergic), whereas shorter i3 loops are found in the photoreceptor rhodopsin or -adrenergic receptors (20 -50 amino acids). During the process of signal initiation and termination, several proteins interact with the receptor. The interaction of arrestins with G-protein-coupled receptors is a key component of signal termination (1-5).The arrestin family consists of visual arrestin, -arrestin, arrestin-3, and a cone-specific arrestin termed C-or X-arrestin (6 -11). In vertebrates, visual arrestin interacts with phosphorylated rhodopsin in rod cells to terminate signal propagation by interfering with receptor coupling to transducin. -Arrestin and arrestin-3 are widely expressed and parallel the role of visual arrestin in terms of signal termination for G-proteincoupled receptors other than rhodopsin. The affinity of arrestin binding to G-protein-coupled receptors is increased by receptor phosphorylation and/or activation by agonist. Receptors of this class are phosphorylated to varying degrees by protein kinase A and C as well as kinases specific for the activated conformation of the receptor (G-protein-coupled receptor kinases). The phosphorylation of the receptor by G-protein-coupled receptor kinases and subsequent arrestin binding are intimately associated with receptor desensitization and sequestration (12)(13)(14). Resensitization of the receptor protein involves dissociation of bound arrestin and receptor dephosphorylation.The interaction of receptors with G-proteins, protein kinases, arrestins, and additional entities controlling receptor trafficking apparently involves discrete motifs in cytoplasmic domains of the receptor. The associations of these proteins with the receptor likely occur within a signal transduction complex that may also include various effector m...
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