Synthetic phosphopeptide from rhodopsin sequence induces retinal arrestin binding to photoactivated unphosphorylated rhodopsin

Puig, Jaime; Arendt, Anatol; Tomson, Farol L.; Abdulaeva, Galina V.; Miller, Ron; Hargrave, Paul A.; McDowell, J. Hugh

doi:10.1016/0014-5793(95)00225-x

Cited by 62 publications

(71 citation statements)

References 18 publications

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“…Binding of 162myc163 or 99myc100 to R* was substantially enhanced in the presence of the 7PP (Fig. 5), an effect similar to that previously reported for wild-type arrestin (20). Binding of 162myc163 to R* was inhibited by the anti-Myc antibody, whereas that of 99myc100 was not affected.…”

Section: Resultssupporting

confidence: 86%

“…The TPCK-treated trypsin was from Sigma. Bovine arrestin and bovine rod cell outer segment (ROS) membranes were prepared as described previously (20).…”

Section: Methodsmentioning

confidence: 99%

“…6, lanes 11 and 12). Previous studies have shown that heparin and other negatively charged ligands such as light-activated phosphorylated rhodopsin or the synthetic 7PP are able to bind arrestin and induce a conformational change in the molecule (20,26,31,38). Although it has been determined that the conformational changes induced by these ligands may not be identical, an enhanced susceptibility to proteolysis of arrestin C terminus is a common effect (38).…”

Section: Fig 2 Binding Of Wild-type and Myc-tagged Arrestin Mutantsmentioning

confidence: 99%

“…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). Additionally, charge reversal or neutralization by mutagenesis of Arg-175 in arrestin produces mutants that are able to bind to light-activated rhodopsin in its unphosphorylated state, as does a splice variant of arrestin that is truncated at its C terminus (21)(22)(23).…”

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confidence: 99%

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Insertional Mutagenesis and Immunochemical Analysis of Visual Arrestin Interaction with Rhodopsin

Dinculescu

McDowell

Amici

et al. 2002

Journal of Biological Chemistry

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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.

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Section: Resultssupporting

confidence: 86%

“…The TPCK-treated trypsin was from Sigma. Bovine arrestin and bovine rod cell outer segment (ROS) membranes were prepared as described previously (20).…”

Section: Methodsmentioning

confidence: 99%

Section: Fig 2 Binding Of Wild-type and Myc-tagged Arrestin Mutantsmentioning

confidence: 99%

mentioning

confidence: 99%

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Insertional Mutagenesis and Immunochemical Analysis of Visual Arrestin Interaction with Rhodopsin

Dinculescu

McDowell

Amici

et al. 2002

Journal of Biological Chemistry

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“…Peptides representing the third and first intracellular loops of rhodopsin were able to inhibit arrestin interactions with light-activated rhodopsin at concentrations of 34 and 1100 M, respectively (35). Furthermore, a synthetic, fully phosphorylated (representing 7 phosphoserine and phosphothreonine residues) 19-mer peptide based on the rhodopsin sequence was demonstrated to induce a conformational change within arrestin as well as enable binding of arrestin to unphosphorylated rhodopsin (36). Based on sulfhydryl reactivity, the conformation of the phosphopeptidebound arrestin was suggested to be different from that of the phosphorylation-independent visual arrestin mutant R175Q (37).…”

Section: Discussionmentioning

confidence: 99%

Arrestin Variants Display Differential Binding Characteristics for the Phosphorylated N-Formyl Peptide Receptor Carboxyl Terminus

Potter¹,

Key²,

Gurevich³

et al. 2002

Journal of Biological Chemistry

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The phosphorylation-dependent binding of arrestins to cytoplasmic domains of G protein-coupled receptors (GPCRs) is thought to be a crucial step in receptor desensitization. In some GPCR systems, arrestins have also been demonstrated to be involved in receptor internalization, resensitization, and the activation of signaling cascades. The objective of the current study was to examine binding interactions of members of the arrestin family with the formyl peptide receptor (FPR), a member of the GPCR family of receptors. Peptides representing the unphosphorylated and phosphorylated carboxyl terminus of the FPR were synthesized and bound to polystyrene beads via a biotin/ streptavidin interaction. Using fluorescein-conjugated arrestins, binding interactions between arrestins and the bead-bound FPR carboxyl terminus were analyzed by flow cytometry. Arrestin-2 and arrestin-3 bound to theFPRcarboxyl-terminalpeptideinaphosphorylationdependent manner, with K d values in the micromolar range. Binding of visual arrestin, which binds rhodopsin with high selectivity, was not observed. Arrestin-2-(1-382) and arrestin-3-(1-393), truncated mutant forms of arrestin that display phosphorylation-independent binding to intact receptors, were also observed to bind the bead-bound FPR terminus in a phosphorylationdependent manner, but with much greater affinity than the full-length arrestins, yielding K d values in the 5-50 nM range. Two additional arrestin mutants, which are full-length but display phosphorylation-independent binding to intact GPCRs, were evaluated for their binding affinity to the FPR carboxyl terminus. Whereas the single point mutant, arrestin-2 R169E, displayed an affinity similar to that of the full-length arrestins, the triple point mutant, arrestin-2 I386A/ V387A/F388A, displayed an affinity more similar to that of the truncated forms of arrestin. The results suggest that the carboxyl terminus of arrestin is a critical determinant in regulating the binding affinity of arrestin for the phosphorylated domains of GPCRs.G protein-coupled receptors (GPCRs) 1 are the largest family of cellular surface receptors, the mediators of numerous physiological responses, and the targets of ϳ50% of therapeutic drugs (1). Their activation occurs through the binding of specific ligands to the extracellular face of the receptor, whereas cellular activation is mediated through interactions with cytoplasmic proteins, such as G proteins. Following ligand-mediated activation, GPCRs are the target of regulatory mechanisms aimed initially at terminating and then at reinitiating the signaling capacity of the cell. Furthermore, GPCR processing has also been shown to be important in the activation of Src and certain MAP kinase pathways (2). The protein arrestin was first characterized as a protein involved in GPCR desensitization following receptor phosphorylation, but has recently been demonstrated to play multiple roles in receptor function, including acting as an adapter for clathrin-mediated endocytosis and a scaffolding protein for...

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Structure and Function of Activated Rhodopsin

Hofmann

Jäger

Ernst

1995

Israel Journal of Chemistry

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Abstract. After cis/trans isomerization of retinal and early photoproducts, activated rhodopsin (R*) develops signaling states for different proteins in a time-ordered sequence. Rhodopsin kinase binds all Meta forms, including the early Meta I, while interaction with transducin (G,) or arrestin requires the deprotonated Schiff base form, Meta II (MIl). G, recognizes a specific conformation, termed MIl b , which arises from an additional, spectrally silent conversion, linked to proton uptake. Collisional coupling with the GDP-bound G, holoprotein induces the release of GDP and formation of a stable R*-G, complex, in which the nucleotide binding site of G, is empty. The empty site complex, once formed, remains stable, even if the retinal is re-isomerized to the cis configuration. The cytoplasmic surface of rhodopsin appears to provide the majority of interaction sites for other proteins. Physical analyses and mutagenesis have emphasized the loops connecting helices CID and ElF of the seven-helix structure. Any interaction between R* and G, depends on a conserved charge pair at the interface between the helix C and the adjacent loop CD. Replacements or deletions in loops CD and EF were found to cause more specific functional defects, including slow release of GDP or failure of GTP-induced complex dissociation. In the dark, signaling states with low activity can be generated by reversible binding of all-trans-retinal to opsin. These lightindependent signaling states are different from the Meta forms.

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Synthetic phosphopeptide from rhodopsin sequence induces retinal arrestin binding to photoactivated unphosphorylated rhodopsin

Cited by 62 publications

References 18 publications

Insertional Mutagenesis and Immunochemical Analysis of Visual Arrestin Interaction with Rhodopsin

Insertional Mutagenesis and Immunochemical Analysis of Visual Arrestin Interaction with Rhodopsin

Arrestin Variants Display Differential Binding Characteristics for the Phosphorylated N-Formyl Peptide Receptor Carboxyl Terminus

Structure and Function of Activated Rhodopsin

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