Role for the Regulator of G-Protein Signaling Homology Domain of G Protein-Coupled Receptor Kinases 5 and 6 in β2-Adrenergic Receptor and Rhodopsin Phosphorylation
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 beta 2AR 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 IC50 of similar to 30 mu M. 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.
机构:
Univ Michigan, Dept Pharmacol, Inst Life Sci, Ann Arbor, MI 48109 USA
Univ Texas Austin, Dept Chem & Biochem, Inst Cellular & Mol Biol, Austin, TX 78712 USAUniv Michigan, Dept Pharmacol, Inst Life Sci, Ann Arbor, MI 48109 USA
Singh, Puja
;
Wang, Benlian
论文数: 0引用数: 0
h-index: 0
机构:
Case Western Reserve Univ, Sch Med, Ctr Prote & Mass Spectrometry, Cleveland, OH 44106 USAUniv Michigan, Dept Pharmacol, Inst Life Sci, Ann Arbor, MI 48109 USA
Wang, Benlian
;
Maeda, Tadao
论文数: 0引用数: 0
h-index: 0
机构:
Case Western Reserve Univ, Sch Med, Dept Pharmacol, Cleveland, OH 44106 USAUniv Michigan, Dept Pharmacol, Inst Life Sci, Ann Arbor, MI 48109 USA
Maeda, Tadao
;
Palczewski, Krzysztof
论文数: 0引用数: 0
h-index: 0
机构:
Case Western Reserve Univ, Sch Med, Dept Pharmacol, Cleveland, OH 44106 USAUniv Michigan, Dept Pharmacol, Inst Life Sci, Ann Arbor, MI 48109 USA
Palczewski, Krzysztof
;
Tesmer, John J. G.
论文数: 0引用数: 0
h-index: 0
机构:
Univ Michigan, Dept Pharmacol, Inst Life Sci, Ann Arbor, MI 48109 USAUniv Michigan, Dept Pharmacol, Inst Life Sci, Ann Arbor, MI 48109 USA
机构:
Thomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USAThomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USA
Thiyagarajan, MM
;
Stracquatanio, RP
论文数: 0引用数: 0
h-index: 0
机构:
Thomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USAThomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USA
Stracquatanio, RP
;
Pronin, AN
论文数: 0引用数: 0
h-index: 0
机构:
Thomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USAThomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USA
Pronin, AN
;
Evanko, DS
论文数: 0引用数: 0
h-index: 0
机构:
Thomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USAThomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USA
Evanko, DS
;
Benovic, JL
论文数: 0引用数: 0
h-index: 0
机构:
Thomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USAThomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USA
Benovic, JL
;
Wedegaertner, PB
论文数: 0引用数: 0
h-index: 0
机构:
Thomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USAThomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USA
机构:
Univ Michigan, Dept Pharmacol, Inst Life Sci, Ann Arbor, MI 48109 USA
Univ Texas Austin, Dept Chem & Biochem, Inst Cellular & Mol Biol, Austin, TX 78712 USAUniv Michigan, Dept Pharmacol, Inst Life Sci, Ann Arbor, MI 48109 USA
Singh, Puja
;
Wang, Benlian
论文数: 0引用数: 0
h-index: 0
机构:
Case Western Reserve Univ, Sch Med, Ctr Prote & Mass Spectrometry, Cleveland, OH 44106 USAUniv Michigan, Dept Pharmacol, Inst Life Sci, Ann Arbor, MI 48109 USA
Wang, Benlian
;
Maeda, Tadao
论文数: 0引用数: 0
h-index: 0
机构:
Case Western Reserve Univ, Sch Med, Dept Pharmacol, Cleveland, OH 44106 USAUniv Michigan, Dept Pharmacol, Inst Life Sci, Ann Arbor, MI 48109 USA
Maeda, Tadao
;
Palczewski, Krzysztof
论文数: 0引用数: 0
h-index: 0
机构:
Case Western Reserve Univ, Sch Med, Dept Pharmacol, Cleveland, OH 44106 USAUniv Michigan, Dept Pharmacol, Inst Life Sci, Ann Arbor, MI 48109 USA
Palczewski, Krzysztof
;
Tesmer, John J. G.
论文数: 0引用数: 0
h-index: 0
机构:
Univ Michigan, Dept Pharmacol, Inst Life Sci, Ann Arbor, MI 48109 USAUniv Michigan, Dept Pharmacol, Inst Life Sci, Ann Arbor, MI 48109 USA
机构:
Thomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USAThomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USA
Thiyagarajan, MM
;
Stracquatanio, RP
论文数: 0引用数: 0
h-index: 0
机构:
Thomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USAThomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USA
Stracquatanio, RP
;
Pronin, AN
论文数: 0引用数: 0
h-index: 0
机构:
Thomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USAThomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USA
Pronin, AN
;
Evanko, DS
论文数: 0引用数: 0
h-index: 0
机构:
Thomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USAThomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USA
Evanko, DS
;
Benovic, JL
论文数: 0引用数: 0
h-index: 0
机构:
Thomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USAThomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USA
Benovic, JL
;
Wedegaertner, PB
论文数: 0引用数: 0
h-index: 0
机构:
Thomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USAThomas Jefferson Univ, Kimmel Canc Ctr, Dept Microbiol & Immunol, Philadelphia, PA 19107 USA