Sulfhydryl Reactivity Demonstrates Different Conformational States for Arrestin, Arrestin Activated by a Synthetic Phosphopeptide, and Constitutively Active Arrestin

McDowell, J. Hugh; Wc, Smith; Rl, Miller; Mp, Popp; Arendt, Anatol; Abdulaeva, Galina V.; Pa, Hargrave

doi:10.1021/bi990175p

Cited by 30 publications

(23 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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). It has also been shown that glutamic acid residues cannot be substituted for the phosphorylated amino acids on this peptide (38).…”

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

View full text Add to dashboard Cite

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...

show abstract

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

View full text Add to dashboard Cite

show abstract

“…The fully phosphorylated form was chosen because multiple phosphorylation was observed in vivo (6,8), and both serine and threonine residues have been shown to be required for activation of the action of arrestin (5, 24 -26). 7PP inhibited phototransduction from photoactivated unphosphorylated rhodopsin in the presence of arrestin, based on the phosphodiesterase assay (27). It also induced conformational changes in arrestin typically seen upon interaction of arrestin with native phosphorylated rhodopsin (14).…”

Section: -Phospho-rh-(330 -348) Peptide Mimics Phosphorylated Cmentioning

confidence: 98%

Conformational Changes in the Phosphorylated C-terminal Domain of Rhodopsin during Rhodopsin Arrestin Interactions

Kisselev

Downs

McDowell³

et al. 2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Phosphorylation of activated G-protein-coupled receptors and the subsequent binding of arrestin mark major molecular events of homologous desensitization. In the visual system, interactions between arrestin and the phosphorylated rhodopsin are pivotal for proper termination of visual signals. By using high resolution proton nuclear magnetic resonance spectroscopy of the phosphorylated C terminus of rhodopsin, represented by a synthetic 7-phosphopolypeptide, we show that the arrestin-bound conformation is a well ordered helixloop structure connected to rhodopsin via a flexible linker. In a model of the rhodopsin-arrestin complex, the phosphates point in the direction of arrestin and form a continuous negatively charged surface, which is stabilized by a number of positively charged lysine and arginine residues of arrestin. Opposite to the mostly extended structure of the unphosphorylated C-terminal domain of rhodopsin, the arrestin-bound C-terminal helix is a compact domain that occupies a central position between the cytoplasmic loops and occludes the key binding sites of transducin. In conjunction with other binding sites, the helix-loop structure provides a mechanism of shielding phosphates in the center of the rhodopsin-arrestin complex and appears critical in guiding arrestin for high affinity binding with rhodopsin.Following activation by a variety of sensory stimuli, such as hormones, neurotransmitters, or light, G-protein-coupled receptors (GPCRs) 1 are deactivated by multiple phosphorylations and subsequent binding of a regulatory protein arrestin (1, 2). Deactivation of the active receptor is obligatory and ensures the quantum character of the response to an extracellular signal. GPCR kinases (GRKs) and arrestin proteins are receptor-specific but share universal mechanisms of action, with prototypical rhodopsin kinase and visual arrestin involved in the termination of visual signal transduction (3, 4). Quenching of the photoresponse in the retinal photoreceptor cells proceeds through phosphorylation of multiple serine and threonine residues at the C terminus of light-activated rhodopsin by GRK1 and high affinity binding of visual arrestin. In vitro studies have implicated all seven serine and threonine residues within the Rh-(334 -343) C-terminal stretch as possible substrates of the phosphorylation reaction. The question of the exact number and position of residues phosphorylated in vivo, however, remains uncertain possibly due to the inherent difficulties of controlling dephosphorylation, different kinetics of phosphorylation/dephosphorylation at individual residues, possible contribution of kinases other than GRK1, and other secondary factors. Either Ser-334 (5) or Ser-343 (6) is phosphorylated initially. The majority of studies provides strong evidence, however, that multiple phosphorylation is required for reproducible deactivation (5-8). In order to circumvent the current uncertainty about the exact sequence of phosphorylation events, we have used a model synthetic peptide phosphorylated at all sev...

show abstract

“…Much evidence, including mutagenesis and in vitro biochemical and biophysical characterizations, suggests that visual arrestin undergoes substantial conformational changes as it binds to light-activated, phosphorylated rhodopsin (93)(94)(95). The X-ray structures of bovine visual arrestin and β-arrestin1 in the basal inactive state indicate that arrestin is an elongated molecule with two domains (N-and C-domain), connected through a 12-residue linker region (96)(97)(98)(99).…”

Section: Activation Of β-Arrestin: Conformational Changesmentioning

confidence: 99%

β-Arrestins and Cell Signaling

DeWire

Ahn

Lefkowitz

et al. 2007

Annu. Rev. Physiol.

1,282

992

View full text Add to dashboard Cite

Upon their discovery, β-arrestins 1 and 2 were named for their capacity to sterically hinder the G protein coupling of agonist-activated seven-transmembrane receptors, ultimately resulting in receptor desensitization. Surprisingly, recent evidence shows that β-arrestins can also function to activate signaling cascades independently of G protein activation. By serving as multiprotein scaffolds, the β-arrestins bring elements of specific signaling pathways into close proximity. β-Arrestin regulation has been demonstrated for an everincreasing number of signaling molecules, including the mitogenactivated protein kinases ERK, JNK, and p38 as well as Akt, PI3 kinase, and RhoA. In addition, investigators are discovering new roles for β-arrestins in nuclear functions. Here, we review the signaling capacities of these versatile adapter molecules and discuss the possible implications for cellular processes such as chemotaxis and apoptosis.

show abstract

Sulfhydryl Reactivity Demonstrates Different Conformational States for Arrestin, Arrestin Activated by a Synthetic Phosphopeptide, and Constitutively Active Arrestin

Cited by 30 publications

References 12 publications

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

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

Conformational Changes in the Phosphorylated C-terminal Domain of Rhodopsin during Rhodopsin Arrestin Interactions

β-Arrestins and Cell Signaling

Contact Info

Product

Resources

About