Transducin-α C-terminal mutations prevent activation by rhodopsin: A new assay using recombinant proteins expressed in cultured cells

Abstract

We have measured the activation by recombinant rhodopsin of the α-subunit (α(t)) of retinal transducin (G(t), also recombinant) using a new assay. Cultured cells are transiently transfected with DNAs encoding opsin and the three subunits of G(t) (α(t), β1 and γ1). In the microsomes of these cells, incubated with 11-cis-retinal, light causes the rapid activation of G(t), as measured by the ability of GTPγS to protect α(t) fragments from proteolytic degradation. The activation of G(t) is also observed when all-trans-retinal is added to microsomes under constant illumination. Activation depends on both opsin and retinal. Opsin mutants with known defects in activating G(t) show similar defects in this assay. α(t) mutations that mimic the corresponding mutations in the α-subunit of G(t) also produce qualitatively similar effects in this assay. As a first step in a strategy aimed at exploring the relationships between structure and function in the interactions of receptors with G proteins, we tested mutant α(t) proteins with alanine substituted for each of the 10 amino acids at the C-terminus, a region known to be crucial for interactions with rhodopsin. Alanine substitution at four positions moderately (K341) or severely (L344, G348, L349) impairs the susceptibility of α(t) to activation by rhodopsin. All four mutants retain their ability to be activated by AIF4-. Two other substitutions (N343 and F350) resulted in very mild defects, while substitutions at the remaining four positions (E342, K345, D346 and C347) had no effect. In combination with previous observations, these results constrain models of the interaction of the C-terminus of α(t) with rhodopsin.