RHODOPSIN MUTANTS DISCRIMINATE SITES IMPORTANT FOR THE ACTIVATION OF RHODOPSIN KINASE AND G(T)

The cytoplasmic loops of rhodopsin, the rod cell photoreceptor, play i mportant regulatory roles in the activation of both rhodopsin kinase a nd the rod cell G protein, G(t). A number of studies have identified d omains in rhodopsin that are important for the activation of G(t). How ever, less is known concerning the cytoplasmic regions that regulate p hosphorylation of the photoreceptor by rhodopsin kinase, To identify r egions that participate in these processes, a series of alanine mutati ons were generated in the three cytoplasmic loops of rhodopsin and tra nsiently expressed in HEK-293 cells, Membranes prepared from these cel ls were reconstituted with the opsin chromophore, Il-cis-retinal, and characterized for their ability to undergo light dependent phosphoryla tion by rhodopsin kinase and to catalyze GTP gamma S (guanosine 5'-O-( 3-thiotriphosphate)) binding to G(t). We have identified mutants that fall into three distinct categories: 1) those that show altered phosph orylation but normal C-t activation, such as T62A/V63A/Q64A and R147A/ F148A/G149A in Loops I and II, respectively; 2) mutants that have redu ced ability to activate G(t) but are phosphorylated normally, includin g T242A/T243A and V250A/T251A/R252A in Loop III; and 3) mutants that a ffect both phosphorylation and G(t) activation, including A233G/A234G/ A235G and A233N/A234N/A235N in Loop III, The use of these two assays i n parallel have allowed us to distinguish the presence of distinct fun ctional domains within the cytoplasmic loops which are specific for in teraction with rhodopsin kinase or G(t).