Phosphorylation modulates the affinity of light-activated rhodopsin for G protein and arrestin

Reduced effector activity and binding of arrestin are widely accepted conse quences of GPCR phosphorylation. However, the effect of receptor multiphosp horylation on G protein activation and arrestin binding parameters has not previously been quantitatively examined. We have found receptor phosphoryla tion to alter both G protein and arrestin binding constants for light-activ ated rhodopsin in proportion to phosphorylation stoichiometry. Rod disk mem branes containing different average receptor phosphorylation stoichiometrie s were combined with G protein or arrestin, and titrated with a series of b rief light flashes. Binding of G(t) or arrestin to activated rhodopsin augm ented 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 co nstant of each to MII were determined using a nonlinear least-squares (Simp lex) reaction model analysis of the titration data. The binding affinity of phosphorylated MII for G, decreased while that for arrestin increased with each added phosphate, G(t) binds more tightly to Mn at phosphorylation lev els less than or equal to two phosphates per rhodopsin; at higher phosphory lation levels, arrestin binding is favored. However, arrestin was found to bind much more slowly than G(t) at all phosphorylation levels, perhaps allo wing time for phosphorylation to gradually reduce receptor-G protein intera ction before arrestin capping of rhodopsin, Sensitivity of the binding cons tants to ionic strength suggests that a strong membrane electrostatic compo nent is involved in both the reduction of G(t) binding and the increase of arrestin binding with increasing rhodopsin phosphorylation.