Cooperativity during multiple phosphorylations catalyzed by rhodopsin kinase: supporting evidence using synthetic phosphopeptides

Pullen, Nicholas; Brown, Neil G.; Sharma, Ram Prakash; Akhtar, Muhammad

doi:10.1021/bi00066a016

Cited by 59 publications

(19 citation statements)

References 23 publications

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“…This result suggests that the introduction of two negative charges enhances the rate of phosphorylation of the remaining serine and threonine residues. A similar cooperativity has been observed by measuring the rate of phosphorylation of rhodopsin containing different numbers of phosphates or the phosphorylation of synthetic peptide substrates corresponding to the rhodopsin COOH terminus (8,(52)(53)(54)(55). Glutamic acid substitution also enhanced the phosphorylation of the formyl-Met-LeuPhe receptor by the G protein-coupled receptor kinase GRK2 (44), implying that cooperativity may be common to the phosphorylation of G protein-coupled receptors by other members of this kinase family.…”

Section: Phosphorylation Of and Arrestin Binding To Rhodopsin Mutantssupporting

confidence: 62%

Rhodopsin Phosphorylation Sites and Their Role in Arrestin Binding

Zhang

Sports

Osawa

et al. 1997

Journal of Biological Chemistry

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Rhodopsin, the rod cell photoreceptor, undergoes rapid desensitization upon exposure to light, resulting in uncoupling of the receptor from its G protein, transducin (G t ). Phosphorylation of serine and threonine residues located in the COOH terminus of rhodopsin is the first step in this process, followed by the binding of arrestin. In this study, a series of mutants was generated in which these COOH-terminal phosphorylation substrate sites were substituted with alanines. These mutants were expressed in HEK-293 cells and analyzed for their ability to be phosphorylated by rhodopsin kinase and to bind arrestin. The results demonstrate that rhodopsin kinase can efficiently phosphorylate other serine and threonine residues in the absence of the sites reported to be the preferred substrates for rhodopsin kinase. A correlation was observed between the level of rhodopsin phosphorylation and the amount of arrestin binding to these mutants. However, mutants T340A and S343A demonstrated a significant reduction in arrestin binding even though the level of phosphorylation was similar to that of wild-type rhodopsin. Substitution of Thr-340 and Ser-343 with glutamic acid residues (T340E and S343E, respectively) was not sufficient to promote the binding of arrestin in the absence of phosphorylation by rhodopsin kinase. When S343E was phosphorylated, its ability to bind arrestin was similar to that of wild-type rhodopsin. Surprisingly, arrestin binding to phosphorylated T340E did not increase to the level observed for wild-type rhodopsin. These results suggest that 2 amino acids, Thr-340 and Ser-343, play important but distinct roles in promoting the binding of arrestin to rhodopsin.Receptor desensitization is a critical process in the regulation of G protein-coupled receptor signaling pathways. Serine and threonine residues located either in the COOH terminus or in the third cytoplasmic loop of these receptors serve as substrates for phosphorylation by members of the G protein-coupled receptor kinase family. The G protein-coupled receptor kinases are unique serine/threonine kinases that phosphorylate only the ligand-activated form of G protein-coupled receptors (1, 2). Phosphorylation is followed by the binding of arrestin to the receptor, resulting in rapid termination of G protein activation (3-5). The physiological importance of rapid receptor desensitization has been demonstrated directly in studies of the visual signal transduction system, in which expression of a truncated form of rhodopsin missing the phosphorylation sites and a selective reduction in the levels of arrestin both lead to extended rhodopsin activity (6, 7).Rhodopsin, the photoreceptor of the rod cell, has been used extensively as a model for investigating the regulation of G protein-coupled receptor desensitization (4). As many as 7 serines and threonines located in the COOH terminus of rhodopsin are substrates in vitro for rhodopsin kinase, the rod cell G protein-coupled receptor kinase, when rhodopsin is activated by light (8). However, several studie...

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Section: Phosphorylation Of and Arrestin Binding To Rhodopsin Mutantssupporting

confidence: 62%

Rhodopsin Phosphorylation Sites and Their Role in Arrestin Binding

Zhang

Sports

Osawa

et al. 1997

Journal of Biological Chemistry

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“…As reported for GRKI, GRK2 and GRK5, GRKs preferentially phosphorylate different SeriThr residues, using photolyzed rhodopsin or /%adrenergic receptor as substrates Fredericks et al, 1996). Multiple phosphorylation might be sequential, with a neighboring hydroxyl-containing amino acid adjoining the primary site of phosphorylation (Pullen et al, 1993;Pullen and Akhtar, 1994;Ohguro et al, 1993Ohguro et al, , 1996. This hierarchical phosphorylation scheme was also proposed for the phosphorylation of the N-formyl peptide receptor by GRK2 (Prossnitz et al, 1995).…”

Section: Gpcrs* Are Substrates For Grks: Two Mechanistic Models Of Grmentioning

confidence: 84%

GTP‐Binding–Protein‐Coupled Receptor Kinases Two Mechanistic Models

Palczewski

1997

European Journal of Biochemistry

108

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Six vertebrate protein kinases (G-protein-coupled receptor kinases; GRKs) that regulate the function of G-protein-coupled receptors (CPCRs) were recently cloned ; several distinct properties set them apart from conventional second-messenger regulated protein kinases. It appears that GRKs bind GPCR" through two separate sites: a high-affinity site, which involves intracellular loops of the activated receptor, and the lower-affinity site, encompassing the phosphorylation region. The high-affinity interaction may involve complementary structural elements of GRKs and GPCRs" rather than precise amino acid alignment, thus allowing broad and overlapping specificities of these kinases, in spite of differences in the sequences of GPCRs. In addition, GRK structures are modified by several posttranslational modifications, including phosphorylation, autophosphorylation, prenylation, carboxymethylation, and palmitoylation, probably affecting properties of these enzymes. While GRKs phosphorylate and inactivate receptor molecules which are engaged in G-protein activation, controversy surrounds whether GRKs might be activated and phosphorylate unstimulated GPCRs, leading to a desensitization of a larger population of the receptors. In this review, mechanistic aspects of GPCR" phosphorylation related to the distinct properties, regulation and modes of action of GRKs are described.

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“…The sojourn time of in state , is taken as an exponentially distributed random variable with mean . The sequences of the phosphorylation rates by RK , the activities of Arr , and the catalytic activities , depend on the underlying biochemistry, and vary with phosphorylation levels [26], [36].…”

Section: Methodsmentioning

confidence: 99%

Kinetics of Rhodopsin Deactivation and Its Role in Regulating Recovery and Reproducibility of Rod Photoresponse

et al. 2010

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The single photon response (SPR) in vertebrate phototransduction is regulated by the dynamics of R* during its lifetime, including the random number of phosphorylations, the catalytic activity and the random sojourn time at each phosphorylation level. Because of this randomness the electrical responses are expected to be inherently variable. However the SPR is highly reproducible. The mechanisms that confer to the SPR such a low variability are not completely understood. The kinetics of rhodopsin deactivation is investigated by a Continuous Time Markov Chain (CTMC) based on the biochemistry of rhodopsin activation and deactivation, interfaced with a spatio-temporal model of phototransduction. The model parameters are extracted from the photoresponse data of both wild type and mutant mice, having variable numbers of phosphorylation sites and, with the same set of parameters, the model reproduces both WT and mutant responses. The sources of variability are dissected into its components, by asking whether a random number of turnoff steps, a random sojourn time between steps, or both, give rise to the known variability. The model shows that only the randomness of the sojourn times in each of the phosphorylated states contributes to the Coefficient of Variation (CV) of the response, whereas the randomness of the number of R* turnoff steps has a negligible effect. These results counter the view that the larger the number of decay steps of R*, the more stable the photoresponse is. Our results indicate that R* shutoff is responsible for the variability of the photoresponse, while the diffusion of the second messengers acts as a variability suppressor.

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Cooperativity during multiple phosphorylations catalyzed by rhodopsin kinase: supporting evidence using synthetic phosphopeptides

Cited by 59 publications

References 23 publications

Rhodopsin Phosphorylation Sites and Their Role in Arrestin Binding

Rhodopsin Phosphorylation Sites and Their Role in Arrestin Binding

GTP‐Binding–Protein‐Coupled Receptor Kinases Two Mechanistic Models

Kinetics of Rhodopsin Deactivation and Its Role in Regulating Recovery and Reproducibility of Rod Photoresponse

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