Mechanistic studies on rhodopsin kinase

Brown, Neil G.; Fowles, Charles; Sharma, Ram Prakash; Akhtar, Muhammad

doi:10.1111/j.1432-1033.1992.tb17232.x

Cited by 46 publications

(50 citation statements)

References 43 publications

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“…Although there are, as yet, no known such endogenous soluble substrates for GRK5, Pitcher et al (32) have recently identified tubulin as a soluble substrate for GRK2. Previous evidence demonstrating that agonist-occupied receptors increase GRK phosphorylation of peptide substrates (33)(34)(35) also support the possibility that endogenous substrates could be phosphorylated following agonist stimulation of GPCR activation of GRKs.…”

Section: Figmentioning

confidence: 59%

Regulation of G protein-coupled Receptor Kinase 5 (GRK5) by Actin

Freeman¹,

Cruz²,

Pollard³

et al. 1998

Journal of Biological Chemistry

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G protein-coupled receptor kinases (GRKs) initiate pathways leading to the desensitization of agonist-occupied G-protein-coupled receptors (GPCRs). Here we report that the cytoskeletal protein actin binds and inhibits GRK5. Actin inhibits the kinase activity directly, reducing GRK5-mediated phosphorylation of both membrane-bound GPCRs and soluble substrates. GRK5 binds actin monomers with a K d of 0.6 M and actin filaments with a K d of 0.2 M. Mutation of 6 amino acids near the amino terminus of GRK5 eliminates actin-mediated inhibition of GRK5. Calmodulin has previously been shown to bind to the amino terminus of GRK5 (Pronin, A. N., and Benovic, J. L. (1997) J. Biol. Chem. 272, 3806 -3812) and here we show calmodulin displaces GRK5 from actin. Calmodulin inhibits GRK5-mediated phosphorylation of GPCRs, but not soluble substrates such as casein. Thus in the presence of actin, calmodulin determines the substrate specificity of GRK5 by preferentially allowing phosphorylation of soluble substrates over membrane-bound substrates.G protein-coupled receptor kinases (GRKs) 1 comprise a family of serine-threonine kinases that desensitize G protein coupled receptors (GPCRs) by phosphorylating agonist occupied receptors (1). The GRKs contain a highly conserved catalytic domain flanked by more divergent amino (NH 2 )-and carboxyl (COOH)-terminal regulatory regions. Based on sequence homology outside the catalytic domain, the GRKs are divided into three subfamilies consisting of GRK1 (Rhodopsin kinase); GRK2 (␤-adrenergic receptor kinase 1) and GRK3 (␤-adrenergic receptor kinase 2); and GRK4, -5, and -6. GRK1 and -4 are expressed exclusively in the retina (2) and testis (3, 4), respectively, while the other GRKs are expressed in most cell types and provide a ubiquitous mechanism for the desensitization of GPCRs. Since GPCR desensitization ultimately results in the termination of many important signaling cascades, it is not surprising that phosphorylation of GPCRs by GRKs is highly regulated.Initially regulation of GPCR phosphorylation focused on the agonist-induced conformational changes receptors undergo to become GRK substrates. However, recent evidence demonstrates GRK activity is regulated differentially by protein kinase C phosphorylation, calcium-binding proteins, and lipid targeting to membranes. Various lipid modifications or binding domains determine accessibility to substrates, as reviewed by Freedman et al. (1). GRK2 and GRK5 are protein kinase C substrates. Protein kinase C phosphorylation activates GRK2 activity, while it inhibits GRK5 activity (5). Additionally, calcium-calmodulin regulates the activity of GRK2 and -3 and GRK4, -5, and -6, but the affinity is 10 -20-fold higher for GRK5 than GRK2 (5-8). Notably, calmodulin binding inhibits GRKmediated GPCR phosphorylation without affecting the ability of these kinases to phosphorylate soluble ("model") substrates. In a search for other protein regulators of GRKs, we have identified a novel interaction between GRKs and actin. Herein we characterize this intera...

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Section: Figmentioning

confidence: 59%

Regulation of G protein-coupled Receptor Kinase 5 (GRK5) by Actin

Freeman¹,

Cruz²,

Pollard³

et al. 1998

Journal of Biological Chemistry

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“…GRK-mediated peptide phosphorylation is markedly enhanced in the presence of activated receptors (66)(67)(68).…”

Section: Grk-mediated Phosphorylation Of Gpcr Substrates: the Agonistmentioning

confidence: 99%

“…And what functional consequences does this multisite interaction have for GRK function? Insight into the functional consequences that such multisite interactions may have for GRK activity first came from studies with rhodopsin, when it was demonstrated that GRK1-mediated phosphorylation of a peptide substrate is significantly enhanced (>100-fold) specifically in the presence of activated rhodopsin (66,67). This effect was achieved with an enzymatically digested form of rhodopsin, which lacked the carboxyl-terminal domain residues phosphorylated by GRK1 (66).…”

Section: Grk-mediated Phosphorylation Of Gpcr Substrates: the Agonistmentioning

confidence: 99%

G Protein–coupled Receptor Kinases

Pitcher

Freedman²,

Lefkowitz³

1998

Annu. Rev. Biochem.

1,178

1,035

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G protein-coupled receptor kinases (GRKs) constitute a family of six mammalian serine/threonine protein kinases that phosphorylate agonist-bound, or activated, G protein-coupled receptors (GPCRs) as their primary substrates. GRK-mediated receptor phosphorylation rapidly initiates profound impairment of receptor signaling, or desensitization. This review focuses on the regulation of GRK activity by a variety of allosteric and other factors: agonist-stimulated GPCRs, βγ subunits of heterotrimeric GTP-binding proteins, phospholipid cofactors, the calcium-binding proteins calmodulin and recoverin, posttranslational isoprenylation and palmitoylation, autophosphorylation, and protein kinase C-mediated GRK phosphorylation. Studies employing recombinant, purified proteins, cell culture, and transgenic animal models attest to the general importance of GRKs in regulating a vast array of GPCRs both in vitro and in vivo. CONTENTS

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“…For example, C-terminally truncated P␥ mutants lacking the last 5-10 amino acids have been reported to result in no inhibition (9,22,23) up to 50% inhibition (15) of catalytic activity. There are also reports that inhibition of PDE6 catalysis can occur without an absolute requirement of the extreme C-terminal amino acids (8,(15)(16)(17).…”

Section: The Photoreceptor Cyclic Nucleotide Phosphodiesterase (Pde6)mentioning

confidence: 99%

Structural Requirements of the Photoreceptor Phosphodiesterase γ-Subunit for Inhibition of Rod PDE6 Holoenzyme and for Its Activation by Transducin

Zhang

Skiba²,

Cote³

2010

Journal of Biological Chemistry

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The central enzyme of the visual transduction cascade, cGMP phosphodiesterase (PDE6), is regulated by its ␥-subunit (P␥), whose inhibitory constraint is released upon binding of activated transducin. It is generally believed that the last four or five C-terminal amino acid residues of P␥ are responsible for blocking catalysis. In this paper, we showed that the last 10 C-terminal residues (P␥78 -87) are the minimum required to completely block catalysis. The kinetic mechanism of inhibition by the P␥ C terminus depends on which substrate is undergoing catalysis. We also discovered a second mechanism of P␥ inhibition that does not require this C-terminal region and that is capable of inhibiting up to 80% of the maximal cGMP hydrolytic rate. Furthermore, amino acids 63-70 and/or the intact ␣2 helix of P␥ stabilize binding of C-terminal P␥ peptides by 100-fold. When PDE6 catalytic subunits were reconstituted with portions of the P␥ molecule and tested for activation by transducin, we found that the C-terminal region (P␥63-87) by itself could not be displaced but that transducin could relieve inhibition of certain P␥ truncation mutants. Our results are consistent with two distinct mechanisms of P␥ inhibition of PDE6. One involves direct interaction of the C-terminal residues with the catalytic site. A second regulatory mechanism may involve binding of other regions of P␥ to the catalytic domain, thereby allosterically reducing the catalytic rate. Transducin activation of PDE6 appears to require interaction with both the C terminus and other regions of P␥ to effectively relieve its inhibitory constraint. The photoreceptor cyclic nucleotide phosphodiesterase (PDE6)2 is the central enzyme in the vertebrate visual signaling pathway in rods and cones. Phototransduction is initiated when light induces the isomerization of the 11-cis-retinal chromophore of rhodopsin, which leads to activation of the photoreceptor-specific G-protein, transducin. Transducin binds GTP and releases its activated ␣-subunit (T␣*-GTP) to activate membrane-associated rod PDE6 by relieving the inhibition of the ␥-subunit at the active sites. The activation of PDE6 results in rapid lowering of cGMP levels, closure of cGMP gated ion channels, and hyperpolarization of the cell membrane (1-5).The PDE6 holoenzyme consists of a catalytic dimer of ␣-and ␤-subunits (P␣␤) and two inhibitory ␥-subunits (P␥) that are tightly bound to P␣␤. Considering its small size, the P␥ subunit of rod and cone PDE6 serves a remarkable number of functions (reviewed in Refs. (6 and 7): 1) a primary function of the P␥ subunit is to inhibit catalysis of cGMP by binding to the catalytic domain of PDE6; 2) the P␥ subunit also enhances the binding affinity of cGMP to the regulatory GAF (cGMP-regulated PDEs, certain adenylate cyclases, and the transcription factor Fh1A of bacteria) domain; 3) activated transducin binds to the P␥ subunit, leading to deinhibition of PDE6 at its active site; and 4) during deactivation, the ␥-subunit participates in a protein complex with the RGS9 and o...

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Mechanistic studies on rhodopsin kinase

Cited by 46 publications

References 43 publications

Regulation of G protein-coupled Receptor Kinase 5 (GRK5) by Actin

Regulation of G protein-coupled Receptor Kinase 5 (GRK5) by Actin

G Protein–coupled Receptor Kinases

Structural Requirements of the Photoreceptor Phosphodiesterase γ-Subunit for Inhibition of Rod PDE6 Holoenzyme and for Its Activation by Transducin

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