PMID:20038610

Citation	Baameur, F, Morgan, DH, Yao, H, Tran, TM, Hammitt, RA, Sabui, S, McMurray, JS, Lichtarge, O and Clark, RB (2010) Role for the regulator of G-protein signaling homology domain of G protein-coupled receptor kinases 5 and 6 in beta 2-adrenergic receptor and rhodopsin phosphorylation. Mol. Pharmacol. 77:405-15
Abstract	Phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) is a major mechanism of desensitization of these receptors. GPCR activation of GRKs involves an allosteric site on GRKs distinct from the catalytic site. Although recent studies have suggested an important role of the N- and C-termini and domains surrounding the kinase active site in allosteric activation, the nature of that site and the relative roles of the RH domain in particular remain unknown. Based on evolutionary trace analysis of both the RH and kinase domains of the GRK family, we identified an important cluster encompassing helices 3, 9, and 10 in the RH domain in addition to sites in the kinase domain. To define its function, a panel of GRK5 and -6 mutants was generated and screened by intact-cell assay of constitutive GRK phosphorylation of the beta(2)-adrenergic receptor (beta 2AR), in vitro GRK phosphorylation of light-activated rhodopsin, and basal catalytic activity measured by tubulin phosphorylation and autophosphorylation. A number of double mutations within helices 3, 9, and 10 reduced phosphorylation of the beta2AR and rhodopsin by 50 to 90% relative to wild-type GRK, as well as autophosphorylation and tubulin phosphorylation. Based on these results, helix 9 peptide mimetics were designed, and several were found to inhibit rhodopsin phosphorylation by GRK5 with an IC(50) of approximately 30 microM. In summary, our studies have uncovered previously unrecognized functionally important sites in the regulator of G-protein signaling homology domain of GRK5 and -6 and identified a peptide inhibitor with potential for specific blockade of GRK-mediated phosphorylation of receptors.
Links	PubMed PMC2835418 Online version:10.1124/mol.109.058115
Keywords	Amino Acid Sequence; Cell Line; G-Protein-Coupled Receptor Kinase 5/physiology; G-Protein-Coupled Receptor Kinases/physiology; Humans; Molecular Sequence Data; Phosphorylation/physiology; Protein Structure, Tertiary; RGS Proteins/physiology; Receptors, Adrenergic, beta-2/metabolism; Rhodopsin/metabolism; Sequence Homology, Amino Acid; Signal Transduction/physiology
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
HUMAN:GRK6		GO:0047696: beta-adrenergic receptor kinase activity	ECO:0000315:		F		Figure 7	complete CACAO 6750
HUMAN:GRK6		GO:0047696: beta-adrenergic receptor kinase activity	ECO:0000314:		F		Fig. 3	complete CACAO 6741
HUMAN:GRK6	enables	GO:0047696: beta-adrenergic receptor kinase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
HUMAN:GRK6	enables	GO:0047696: beta-adrenergic receptor kinase activity	ECO:0000315: mutant phenotype evidence used in manual assertion		F		Seeded From UniProt	complete
HUMAN:GRK5		GO:0047696: beta-adrenergic receptor kinase activity	ECO:0000315:		F		Fig 4.	complete CACAO 6735
HUMAN:GRK5	enables	GO:0047696: beta-adrenergic receptor kinase activity	ECO:0000315: mutant phenotype evidence used in manual assertion		F		Seeded From UniProt	complete

References

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