Mechanism of Quenching of Phototransduction

␤-Arrestins are crucial regulators of G-protein coupled receptor (GPCR) signaling, desensitization, and internalization. Despite the long-standing paradigm that agonist-promoted receptor phosphorylation is required for ␤-arrestin2 recruitment, emerging evidence suggests that phosphorylation-independent mechanisms play a role in ␤-arrestin2 recruitment by GPCRs. Several PDZ proteins are known to interact with GPCRs and serve as cytosolic adaptors to modulate receptor signaling and trafficking. Na ؉ /H ؉ exchange regulatory factors (NHERFs) exert a major role in GPCR signaling. By combining imaging and biochemical and biophysical methods we investigated the interplay among NHERF1, ␤-arrestin2, and the parathyroid hormone receptor type 1 (PTHR). We show that NHERF1 and ␤-arrestin2 can independently bind to the PTHR and form a ternary complex in cultured human embryonic kidney cells and Chinese hamster ovary cells. Although NHERF1 interacts constitutively with the PTHR, ␤-arrestin2 binding is promoted by receptor activation. NHERF1 interacts directly with ␤-arrestin2 without using the PTHR as an interface. Fluorescence resonance energy transfer studies revealed that the kinetics of PTHR and ␤-arrestin2 interactions were modulated by NHERF1. These findings suggest a model in which NHERF1 may serve as an adaptor, bringing ␤-arrestin2 into close proximity to the PTHR, thereby facilitating ␤-arrestin2 recruitment after receptor activation.Scaffold proteins play an increasingly recognized role in the regulation and the signal transduction of G protein coupled receptors (GPCRs).2 The interaction of GPCRs with ␤-arrestins results in several distinct effects: (i) signal termination by homologous receptor desensitization, (ii) recruitment of elements of the internalization machinery thereby promoting GPCR endocytosis, and (iii) switching GPCR signaling to nonclassical pathways such as the mitogen-activated protein kinase pathway (1-3). Despite their limited sequence homology, nearly all GPCRs bind to ␤-arrestins, and ␤-arrestin recruitment is considered to be a universal mechanism for GPCR regulation. 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...

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confidence: 99%

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

Klenk

Vetter

Zürn

et al. 2010

“…1 shows two examples of how synthetic peptides derived from the arrestin sequence influence the formation of extra MII from prephosphorylated samples. The arrestin peptide comprising residues 11-30 (arrestin- (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)) reduced the amount of extra MII formed in a concentration-dependent manner (Fig. 1A, compare traces b and c); at 1000 M, arrestin-(11-30) abolished extra MII nearly to the control level without arrestin (compare traces c and e).…”

Section: Light-induced Interaction Between Rhodopsin and Arrestin Ismentioning

confidence: 99%

“…Peptide competition has already been applied in previous studies to map regions of interaction in the receptor-arrestin complex. Studies with peptides from the surface-exposed sequences of rhodopsin suggested a role of different loop structures in the interaction (26). Regarding the interactive domains in arrestin, Hargrave and co-workers (27) have employed a library of peptides covering the entire sequence.…”

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confidence: 99%

Interactions of Metarhodopsin II

Pulvermüller

Schröder

Fischer

et al. 2000

Arrestin blocks the interaction of rhodopsin with the G protein transducin (G t ). To characterize the sites of arrestin that interact with rhodopsin, we have utilized a spectrophotometric peptide competition assay. It is based on the stabilization of the active intermediates metarhodopsin II (MII) and phosphorylated MII by G t and arrestin, respectively (extra MII monitor). The protocol involves native disc membranes and three sets of peptides 10 -30 amino acids in length spanning the arrestin sequence. In the absence of arrestin, not one of the peptides by itself had an effect on the amount of MII formed. However, inhibition of arrestin-dependent extra MII was found for the peptides at residues 11-30 and 51-70 (IC 50 < 100 M) and residues 231-260 (IC 50 < 200 M). A similar pattern of inhibition by arrestin peptides was seen when arrestin was replaced by G t or the farnesylated G t ␥ C-terminal peptide. Only arrestin-(11-30) inhibited MII⅐G t less (IC 50 ‫؍‬ 300 M) than phosphorylated MII⅐arrestin. We interpreted the data by competition of the arrestin peptides for interaction sites at rhodopsin, exposed in the MII conformation and specific for both arrestin and G t . The arrestin sites are located in both the C-and N-terminal domains of the arrestin structure.G protein-coupled receptors enable eukaryotic cells to respond to a large variety of extracellular signals, including hormones, odorants, and light (1). One of the best studied G protein-coupled receptor-initiated signaling pathways is the visual cascade in retinal rods (2). It is initiated by the absorption of a photon in the visual receptor (rhodopsin) and subsequent isomerization of the 11-cis-retinal, which is covalently attached to Lys 296 in the seventh transmembrane helix of the receptor. Via a series of transient intermediates, rhodopsin converts in milliseconds into metarhodopsin II (MII), 1 the form of rhodopsin that interacts with the heterotrimeric G protein transducin (G t ). This interaction initiates the intermolecular transduction of the light signal by catalyzing GDP/GTP exchange in the nucleotide-binding site of the G protein and in turn activates the G protein-coupled effector, a cGMP-specific phosphodiesterase. Hydrolysis of cGMP leads to the closure of plasma membrane cation channels and hyperpolarization of the membrane (3).The deactivation of rhodopsin starts with the binding of a rhodopsin kinase to photoactivated rhodopsin (4). Rapid phosphorylation of the receptor at C-terminal sites (5) increases its affinity for arrestin (6). Biochemical (7) and electrophysiological (8) evidence has been accumulated that visual arrestins deactivate the visual cascade by direct competition with the G protein for the active receptor. However, the mechanism of interaction underlying the quench is still not well understood.UV-visible spectroscopy has shown that, to bind arrestin, the light activation of rhodopsin must proceed up to the MII conformation, in which the Schiff base bond of the retinal to the apoprotein is still intact but deprotonated...

“…Phosphorylation is an important chemical modification that allows the activated receptors to be further recognized by proteins from the arrestin family (5). Arrestins stop the signal transduction quickly by directly preventing the G-protein access to the cytoplasmic loops of activated receptors (6). This leads to a diminished cellular responsiveness to a specific stimulus, a phenomenon known as homologous desensitization.…”

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confidence: 99%

“…Next, visual arrestin binds with high affinity and specificity to light-activated, phosphorylated rhodopsin by a mechanism that is incompletely understood (15,16). Experimental evidence suggests that in addition to phosphate binding residues, arrestin contains elements that enable it to specifically recognize the loops of the photoactivated rhodopsin (17)(18)(19). It has been shown that arrestin is able to recognize the activated, unphosphorylated rhodopsin or a C-terminal truncated form of the activated rhodopsin in the presence of a synthetic fully phosphorylated peptide corresponding to the last 19 amino acids of the C terminus of rhodopsin (20).…”

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confidence: 99%

Insertional Mutagenesis and Immunochemical Analysis of Visual Arrestin Interaction with Rhodopsin

Dinculescu

McDowell

Amici

et al. 2002

Visual arrestin inactivates the phototransduction cascade by specifically binding to light-activated phosphorylated rhodopsin. This study describes the combined use of insertional mutagenesis and immunochemical approaches to probe the structural determinants of arrestin function. Recombinant arrestins with insertions of a 10-amino acid c-Myc tag (EQKLISEEDL) were expressed in yeast and characterized. When the tag was placed on the C terminus after amino acid 399, between amino acids 99 and 100 or between residues 162 and 163, binding to rhodopsin was found to be very similar to that of wild-type arrestin. Two stable mutants with Myc insertions in the 68 -78 loop were also generated. Binding to rhodopsin was markedly decreased for one (72myc73) and completely abolished for the other (77myc78). Limited proteolysis assays using trypsin in the absence or presence of heparin were performed on all mutants and confirmed their overall conformational integrity. Rhodopsin binding to either 162myc163 or 72myc73 arrestins in solution was completely inhibited in the presence of less than a 2-fold molar excess of anti-Myc antibody relative to arrestin. In contrast, the antibody did not block the interaction of the 399myc or 99myc100 arrestins with rhodopsin. These results indicate that an interactive surface for rhodopsin is located on or near the concave region of the N-domain of arrestin.