Targeted Construction of Phosphorylation-independent β-Arrestin Mutants with Constitutive Activity in Cells

Abstract

Arrestin proteins play a key role in the desensitization of G protein-coupled receptors (GPCRs). Recently we proposed a molecular mechanism whereby arrestin preferentially binds to the activated and phosphorylated form of its cognate GPCR. To test the model, we introduced two different types of mutations into ␤-arrestin that were expected to disrupt two crucial elements that make ␤-arrestin binding to receptors phosphorylation-dependent. We found that two ␤-arrestin mutants (Arg 169 3 Glu and Asp 383 3 Ter) (Ter, stop codon) are indeed "constitutively active." In vitro these mutants bind to the agonist-activated ␤ 2 -adrenergic receptor (␤ 2 AR) regardless of its phosphorylation status. When expressed in Xenopus oocytes these ␤-arrestin mutants effectively desensitize ␤ 2 AR in a phosphorylationindependent manner. Constitutively active ␤-arrestin mutants also effectively desensitize ␦ opioid receptor (DOR) and restore the agonist-induced desensitization of a truncated DOR lacking the critical G protein-coupled receptor kinase (GRK) phosphorylation sites. The kinetics of the desensitization induced by phosphorylation-independent mutants in the absence of receptor phosphorylation appears identical to that induced by wild type ␤-arrestin ؉ GRK3. Either of the mutations could have occurred naturally and made receptor kinases redundant, raising the question of why a more complex two-step mechanism (receptor phosphorylation followed by arrestin binding) is universally used.

Platelet-activating Factor-induced Chemokine Gene Expression Requires NF-κB Activation and Ca2+/Calcineurin Signaling Pathways

“…4) raises the interesting possibility that ␤-arrestin could actually provide an inhibitory signal for NF-B activation and CCL2 production. A constitutively active mutant of ␤-arrestin (␤arr-R169E) has been shown to associate with phosphorylation-deficient mutants of a number of G protein-coupled receptors (43)(44)(45). To determine the role of ␤-arrestin on PAFinduced responses, transient transfectants were generated in RBL-2H3 cells coexpressing ⌬ST-PAFR with ␤arr-R169E/ green fluorescent protein conjugate (GFP-␤arr-R169E).…”

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).…”

Formation of a Ternary Complex among NHERF1, β-Arrestin, and Parathyroid Hormone Receptor

“…Rather than binding to a specific motif, ␤-arrestins appear to interact with several parts of the receptor involving the second and third intracellular loops and the C terminus (4 -6). It is generally thought that ␤-arrestin-receptor interaction involves an initial weak interaction of cytosolic ␤-arrestins with an active and phosphorylated receptor conformation, followed by conformational rearrangements of ␤-arrestins that further stabilize the receptor⅐arrestin complex (7,8).…”

“…Rather than binding to a specific motif, ␤-arrestins appear to interact with several parts of the receptor involving the second and third intracellular loops and the C terminus (4 -6). It is generally thought that ␤-arrestin-receptor interaction involves an initial weak interaction of cytosolic ␤-arrestins with an active and phosphorylated receptor conformation, followed by conformational rearrangements of ␤-arrestins that further stabilize the receptor⅐arrestin complex (7,8).The receptor for parathyroid hormone (PTH) and PTH-related protein is involved in regulating calcium homeostasis and bone remodeling (9). Agonist occupancy of the PTH/PTH-related protein receptor (PTHR) leads to G s -mediated stimulation of adenylyl cyclase, resulting in cAMP production and PKA activation, and G q/11 -mediated phosphatidylinositol-specific phospholipase C␤ stimulation, leading to inositol 1,4,5-trisphosphate formation, calcium mobilization, and PKC activation (10 -12).…”