Visual Arrestin Binding to Rhodopsin

Gurevich, Vsevolod V.; Benovic, Jeffrey L.

doi:10.1074/jbc.270.11.6010

Cited by 171 publications

(293 citation statements)

References 21 publications

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“…There are several conserved domains that may be important for receptor recognition, binding and intramolecular reactions ( Figure 5). These domains have been characterized by mutagenesis and generation of chimaeric molecules [107][108][109].…”

Section: Arrestin Familiesmentioning

confidence: 99%

Regulatory mechanisms that modulate signalling by G-protein-coupled receptors

Böhm

Grady

Bunnett

1997

Biochemical Journal

476

322

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The large and functionally diverse group of G-protein-coupled receptors includes receptors for many different signalling molecules, including peptide and non-peptide hormones and neurotransmitters, chemokines, prostanoids and proteinases. Their principal function is to transmit information about the extracellular environment to the interior of the cell by interacting with the heterotrimeric G-proteins, and they thereby participate in many aspects of regulation. Cellular responses to agonists of these receptors are usually rapidly attenuated. Mechanisms of signal attenuation include removal of agonists from the extracellular fluid, receptor desensitization, endocytosis and downregulation. Agonists are removed by dilution, uptake by transporters and enzymic degradation. Receptor desensitization is mediated by receptor phosphorylation by G-protein receptor kinases and second-messenger kinases, interaction of phosphorylated receptors with arrestins and receptor uncoupling from

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Section: Arrestin Familiesmentioning

confidence: 99%

Regulatory mechanisms that modulate signalling by G-protein-coupled receptors

Böhm

Grady

Bunnett

1997

Biochemical Journal

476

322

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“…Protein Expression and Purification-Urea-stripped rod outer segments were prepared as described previously (20). G␤ 1 ␥ 2 (prepared as described in Ref.…”

Section: Methodsmentioning

confidence: 99%

Mutational Analysis of Gβγ and Phospholipid Interaction with G Protein-coupled Receptor Kinase 2

Carman

Barak

Chen

et al. 2000

Journal of Biological Chemistry

Self Cite

101

100

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Agonist-dependent regulation of G protein-coupled receptors is dependent on their phosphorylation by G protein-coupled receptor kinases (GRKs). GRK2 and GRK3 are selectively regulated in vitro by free G␤␥ subunits and negatively charged membrane phospholipids through their pleckstrin homology (PH) domains. However, the molecular binding determinants and physiological role for these ligands remain unclear. To address these issues, we generated an array of site-directed mutants within the GRK2 PH domain and characterized their interaction with G␤␥ and phospholipids in vitro. Mutation of several residues in the loop 1 region of the PH domain, including Lys-567, Trp-576, Arg-578, and Arg-579, resulted in a loss of receptor phosphorylation, likely via disruption of phospholipid binding, that was reversed by G␤␥. Alternatively, mutation of residues distal to the C-terminal amphipathic ␣-helix, including Lys-663, Lys-665, Lys-667, and Arg-669, resulted in decreased responsiveness to G␤␥. Interestingly, mutation of Arg-587 in ␤-sheet 3, a region not previously thought to interact with G␤␥, resulted in a specific and profound loss of G␤␥ responsiveness. To further characterize these effects, two mutants (GRK2(K567E/R578E) and GRK2(R587Q)) were expressed in Sf9 cells and purified. Analysis of these mutants revealed that GRK2(K567E/R578E) was refractory to stimulation by negatively charged phospholipids but bound G␤␥ similar to wild-type GRK2. In contrast, GRK2(R587Q) was stimulated by acidic phospholipids but failed to bind G␤␥. In order to examine the role of phospholipid and G␤␥ interaction in cells, wild-type and mutant GRK2s were expressed with a ␤ 2 -adrenergic receptor (␤ 2 AR) mutant that is responsive to GRK2 phosphorylation (␤ 2 AR(Y326A)). In these cells, GRK2(K567E/R578E) and GRK2(R587Q) were largely defective in promoting agonist-dependent phosphorylation and internalization of ␤ 2 AR(Y326A). Similarly, wild-type GRK2 but not GRK2(K567E/R578E) or GRK2(R587Q) promoted morphinedependent phosphorylation of the -opioid receptor in cells. Thus, we have (i) identified several specific GRK2 binding determinants for G␤␥ and phospholipids, and (ii) demonstrated that G␤␥ binding is the limiting step for GRK2-dependent receptor phosphorylation in cells.Diverse extracellular stimuli are perceived at the plasma membrane by G protein-coupled receptors (GPCRs).1 Agonistoccupied receptors promote the activation and dissociation of the heterotrimeric G protein ␣ and ␤␥ subunits, each of which goes on to regulate various effector molecules thereby producing a physiological response. This process is regulated in an agonist-dependent fashion by a family of G protein-coupled receptor kinases (GRKs), which phosphorylate activated GPCRs promoting binding of a second family of proteins, termed arrestins, which serve to uncouple the GPCR from further G protein activation (1, 2). Arrestin binding also promotes receptor internalization, which facilitates the processes of receptor resensitization and down-regulation (1).In general, all G...

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“…4, B and corresponding image D), suggesting that GRK phosphorylation increases the affinity of the receptor for ␤-arrestin (22). To further characterize the role of GRK phosphorylation in ␤-arrestin translocation, we examined the ability of a GRK phosphorylation-impaired mutant Y326A-␤ 2 AR to support ␤-arrestin redistribution (11,17,23,24). Consistent with its inability to be phosphorylated by endogenous GRKs, the Y326A-␤2AR mutant did not induce ␤arr2-GFP translocation with agonist exposure (Fig.…”

Section: Universal Detection Of G Protein-coupled Receptor Activationmentioning

confidence: 99%

A β-Arrestin/Green Fluorescent Protein Biosensor for Detecting G Protein-coupled Receptor Activation

Barak

Ferguson²,

Zhang

et al. 1997

Journal of Biological Chemistry

419

367

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G protein-coupled receptors (GPCR) represent the single most important drug targets for medical therapy, and information from genome sequencing and genomic data bases has substantially accelerated their discovery. The lack of a systematic approach either to identify the function of a new GPCR or to associate it with a cognate ligand has added to the growing number of orphan receptors. In this work we provide a novel approach to this problem using a ␤-arrestin2/green fluorescent protein conjugate (␤arr2-GFP). It provides a real-time and single cell based assay to monitor GPCR activation and GPCR-G protein-coupled receptor kinase or GPCR-arrestin interactions. Confocal microscopy demonstrates the translocation of ␤arr2-GFP to more than 15 different ligand-activated GPCRs. These data clearly support the common hypothesis that the ␤-arrestin binding of an activated receptor is a convergent step of GPCR signaling, increase by 5-fold the number of GPCRs known to interact with ␤-arrestins, demonstrate that the cytosol is the predominant reservoir of biologically active ␤-arrestins, and provide the first direct demonstration of the critical importance of G protein-coupled receptor kinase phosphorylation to the biological regulation of ␤-arrestin activity and GPCR signal transduction in living cells. The use of ␤arr2-GFP as a biosensor to recognize the activation of pharmacologically distinct GPCRs should accelerate the identification of orphan receptors and permit the optical study of their signal transduction biology intractable to ordinary biochemical methods.The G protein-coupled receptor (GPCR) 1 superfamily is growing rapidly (1-3), creating many new orphan receptors whose properties remain undefined (4 -6). 2 Currently characterized GPCRs display many distinct pharmacologies. For example, they interact with a vast array of ligands and generate intracellular signals by multiple second messenger pathways (4,8,9). Based on work with rhodopsin and the ␤ 2 -adrenergic receptor (␤ 2 AR), it has been postulated that members of the GPCR superfamily desensitize via a common mechanism involving the arresting proteins visual arrestin, ␤-arrestin1 and ␤-arrestin2 (10 -13). However, mainly due to the inherent difficulties of examining the interaction of the components mediating desensitization in their native environment or the need for purified reconstituted systems, this has not been clearly established for many GPCRs. Biochemical studies indicate that arrestins regulate GPCR signal transduction (desensitization) by binding agonist-activated receptors that have been phosphorylated by G protein-coupled receptor kinases (GRKs) (12). While the functional source of arrestin molecules targeted to receptors remains unknown, it is apparent that arrestin binding terminates signaling by interdicting receptor interaction with G proteins (12).To characterize the interaction between ␤-arrestin and different GPCRs and to assess the contribution of GRKs to this process, we examined using confocal microscopy how a green fluorescent protein...

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Visual Arrestin Binding to Rhodopsin

Cited by 171 publications

References 21 publications

Regulatory mechanisms that modulate signalling by G-protein-coupled receptors

Regulatory mechanisms that modulate signalling by G-protein-coupled receptors

Mutational Analysis of Gβγ and Phospholipid Interaction with G Protein-coupled Receptor Kinase 2

A β-Arrestin/Green Fluorescent Protein Biosensor for Detecting G Protein-coupled Receptor Activation

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