Expression, purification, and characterization of the G protein-coupled receptor kinase GRK6.
1994; Elsevier BV; Volume: 269; Issue: 36 Linguagem: Inglês
10.1016/s0021-9258(17)31701-5
ISSN1083-351X
Autores Tópico(s)Nitric Oxide and Endothelin Effects
ResumoG protein-coupled receptor kinases (GRKs), such as rhodopsin kinase and P-adrenergic receptor kinase (PARK), are involved in mediating agonist-specific phosphorylation and desensitization of G protein-coupled receptors.GRKG is the most recently identified member of the GRK family and displays higher homology with GRK5 (70.1% amino acid identity) and ITll (68.5%) compared to PARK (37.4%) and rhodopsin kinase (47.1%).To further characterize GRK6, it has been overexpressed in Sf9 cells and purified to homogeneity by sequential chromatography on SP-Sepharose and heparin-Sepharose columns.GRK6 shares a number of in vitro characteristics with GRK5, including potent inhibition by heparin and dextran sulfate (IC, values of -15 and -7 n ~, respectively), hyperstimulation by polycations, and preference for phosphorylation of non-acidic peptides.Rhodopsin and the &-adrenergic and m2 muscarinic cholinergic receptors serve as stimulus-dependent substrates for GRK6, but with stoichiometries significantly lower than achieved by GRK5 and PARK.Additionally, GRK6 does not undergo significant autophosphorylation even though it contains residues identical to those that are autophosphorylated in GRK5 and rhodopsin kinase.These data extend our knowledge of a growing family of receptor-specific kinases and suggest that GRK6 has a substrate specificity distinct from PARK, rhodopsin kinase, and GRK5.Many transmembrane signal-transducing pathways involve distinct G protein'-coupled receptors.These molecules are able to transmit intracellular signals that arise as a consequence of extracellular ligand binding (i.e.hormones, neurotransmitters, odorants, and light) to the appropriate receptor (1, 2).G protein-coupled receptors regulate a variety of effector molecules such as adenylyl cyclase, cGMP phosphodiesterase, phospholipase C, phospholipase 4, and numerous ion channels.Two of the most extensively studied receptors are the &-adrenergic receptor which mediates catecholamine stimulation of adenylyl cyclase, and the visual light receptor rhodopsin, which facilitates phototransduction in retinal rod cells (3,4).In both systems a rapid diminution of responsiveness or desensitiza-~~ ~
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