Monomeric Wild Type And Constitutively Active Rhodopsin Is Effectively Phosphorylated By Different Grks And Binds Arrestin-1

Abstract

Purpose: : To assess the phosphorylation of monomeric wild type (WT) rhodopsin and constitutively active M257Y mutant by different GRKs and determine relative degree of arrestin binding.

Methods: : Recombinant purified WT and M257Y rhodopsin were reconstituted as monomers into nanodiscs and used for in vitro phosphorylation by purified GRK1, GRK2, and GRK5. The binding of WT arrestin-1 and constitutively active mutants to phosphorylated dark and light-activated (P-Rh*) rhodopsins was measured.

Results: : GRK1, GRK2, and GRK5 transfer 2-3 phosphates per rhodopsin monomer under conditions that yield virtually the same phosphorylation levels of bovine WT rhodopsin in native disc membranes. Thus, for the activation and phosphate transfer GRKs only require the determinants present in a single rhodopsin molecule. High binding of arrestin-1 to P-Rh* does not depend on the GRK that phosphorylated it, showing that all GRKs transfer phosphates to the same or functionally equivalent positions. WT arrestin-1 shows higher binding to dark phosphorylated M257Y than to WT rhodopsin, consistent with constitutive activity of M257Y mutant. Pre-activated arrestin-1-(1-378) and 3A mutants, where the C-tail is deleted or detached from the body of the molecule by triple alanine substitution, demonstrate higher binding to P-Rh* form of both rhodopsins. Moreover, the binding of arrestin-1-3A and (1-378) to dark phosphorylated M257Y reaches ~60% of their binding to P-Rh*, whereas in case of WT rhodopsin it is only ~10% of P-Rh* binding.

Conclusions: : Monomeric WT and mutant rhodopsin is sufficient for effective phosphorylation by all GRKs, demonstrating that GRKs can be effectively activated by monomeric GPCRs. In all cases the phosphorylation promotes high-affinity arrestin-1 binding.

Keywords: protein structure/function • photoreceptors • signal transduction