title: Phosphorylation modulates the affinity of light-activated rhodopsin for G protein and arrestin.
abstract: Reduced effector activity and binding of arrestin are widely accepted consequences of GPCR phosphorylation. However, the effect of receptor multiphosphorylation on G protein activation and arrestin binding parameters has not previously been quantitatively examined. We have found receptor phosphorylation to alter both G protein and arrestin binding constants for light-activated rhodopsin in proportion to phosphorylation stoichiometry. Rod disk membranes containing different average receptor phosphorylation stoichiometries were combined with G protein or arrestin, and titrated with a series of brief light flashes. Binding of G(t) or arrestin to activated rhodopsin augmented the 390 nm MII optical absorption signal by stabilizing MII as MII.G or MII.Arr. The concentration of active arrestin or G(t) and the binding constant of each to MII were determined using a nonlinear least-squares (Simplex) reaction model analysis of the titration data. The binding affinity of phosphorylated MII for G(t) decreased while that for arrestin increased with each added phosphate. G(t) binds more tightly to MII at phosphorylation levels less than or equal to two phosphates per rhodopsin; at higher phosphorylation levels, arrestin binding is favored. However, arrestin was found to bind much more slowly than G(t) at all phosphorylation levels, perhaps allowing time for phosphorylation to gradually reduce receptor-G protein interaction before arrestin capping of rhodopsin. Sensitivity of the binding constants to ionic strength suggests that a strong membrane electrostatic component is involved in both the reduction of G(t) binding and the increase of arrestin binding with increasing rhodopsin phosphorylation.
authors.affiliation: Department of Biochemistry and Biophysics, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-6059, USA.
authors.forename: S K
authors.initials: SK
authors.lastname: Gibson
authors.forename: J H
authors.initials: JH
authors.lastname: Parkes
authors.forename: P A
authors.initials: PA
authors.lastname: Liebman
mesh_terms: D000465:Algorithms; D000818:Animals; D019393:Arrestin / Q000378:metabolism*; D002417:Cattle; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D009994:Osmolar Concentration; D010766:Phosphorylation; D011485:Protein Binding; D012243:Rhodopsin / Q000031:analogs & derivatives / Q000378:metabolism*; D055672:Static Electricity
chemical_list: D019393:Arrestin; C013110:metarhodopsins; D012243:Rhodopsin; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go