Sk. Gibson et al., Phosphorylation modulates the affinity of light-activated rhodopsin for G protein and arrestin, BIOCHEM, 39(19), 2000, pp. 5738-5749
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.