Conservation of the Phosphate-sensitive Elements in the Arrestin Family of Proteins

Abstract

Arrestins play a key role in the homologous desensitization of G protein-coupled receptors (GPCRs). These cytosolic proteins selectively bind to the agonist-activated and GPCR kinase-phosphorylated forms of the GPCR, precluding its further interaction with the G protein. Certain mutations in visual arrestin yield "constitutively active" proteins that bind with high affinity to the light-activated form of rhodopsin without requiring phosphorylation. The crystal structure of visual arrestin shows that these activating mutations perturb two groups of intramolecular interactions that keep arrestin in its basal (inactive) state. Here we introduced homologous mutations into arrestin2 and arrestin3 and found that the resulting mutants bind to the ␤ 2 -adrenoreceptor in vitro in a phosphorylation-independent fashion. The same mutants effectively desensitize both the ␤ 2 -adrenergic and ␦-opioid receptors in the absence of receptor phosphorylation in Xenopus oocytes. Moreover, the arrestin mutants also desensitize the truncated ␦-opioid receptor from which the C terminus, containing critical phosphorylation sites, has been removed. Conservation of the phosphate-sensitive hot spots in nonvisual arrestins suggests that the overall fold is similar to that of visual arrestin and that the mechanisms whereby receptor-attached phosphates drive arrestin transition into the active binding competent state are conserved throughout the arrestin family of proteins.Signaling by various members of the superfamily of G protein-coupled receptors (GPCRs) 1 is attenuated by a uniform two-step mechanism (1). First, the agonist-activated receptor that catalyzes GDP/GTP exchange on G proteins is specifically phosphorylated by a G protein-coupled receptor kinase (GRK). An arrestin protein then binds to the active phosphoreceptor, which makes further G protein interaction impossible by simple steric exclusion (2). Because of the high affinity of nonvisual arrestins 2 for various components of the trafficking machinery (3-5), the arrestin-receptor complex is then internalized. Arrestin binding also serves to switch GPCR signaling from G proteins to various mitogen-activated protein kinases (6 -8). The loss of active receptor conformation in the endosomes (presumably due to ligand dissociation) facilitates arrestin dissociation, rendering the phosphoreceptor accessible to protein phosphatases. The dephosphorylated receptor can then be recycled back to the plasma membrane (9).Perfectly timed binding and dissociation of arrestins, which are ensured by their remarkable selectivity for the phosphorylated/activated form of their cognate receptors, are equally important for high fidelity of this quenching mechanism. Destabilization of certain intramolecular interactions that support the basal (inactive) arrestin conformation by the activated phosphoreceptor is the mechanistic basis of arrestin selectivity (10,15,19,20). The visual arrestin residues involved in the stabilization of its basal state have been identified by extensive mutagenesis (10 -15,...

Mu Opioid Receptor Activation of ERK1/2 Is GRK3 and Arrestin Dependent in Striatal Neurons

“…The dominant positive arrestin3-(R170E) has previously been reported to bind to agonist-occupied receptors and mediate desensitization independent of receptor phosphorylation (10,33). When the arrestin3-(R170E) mutant and a phosphorylation insensitive delta opioid receptor were expressed, desensitization of the receptor was similar to the wild type (33).…”

Conformation of receptor-bound visual arrestin

“…Among the four vertebrate subtypes, arrestin-1 shows the highest receptor specificity and selectivity for P-Rh* (50). However, the mechanism of arrestin activation by GPCRs is conserved in all subtypes (17,18,53), suggesting that receptor binding induces similarly small domain movement in nonvisual arrestins, which leaves a large portion of the molecule essentially unchanged. This can explain why many nonreceptor signaling proteins bind comparably to free and GPCR-associated arrestins (53).…”

“…Simultaneous engagement of both sensors, which only P-Rh* can achieve, triggers a global conformational change, allowing arrestin-1 transition to a high-affinity receptor-binding state. The activation mechanism appears to be conserved in nonvisual arrestins (17,18) that initiate a second round of signaling upon receptor binding. Thus, the arrestin-receptor complex serves as a nucleus of a signalosome (19), where the conformation of the receptor-bound arrestin apparently determines its interactions with multiple signaling proteins (20).…”

The Differential Engagement of Arrestin Surface Charges by the Various Functional Forms of the Receptor

“…11). Only the "phosphate sensor" in arrestin has been characterized extensively (6,8,9,(12)(13)(14)(15). It was shown that receptor-attached phosphates must break the salt bridge between Arg-175 and Asp-296 to destabilize the polar core and make high affinity arrestin binding possible (9).…”