TRANSDUCIN-ALPHA C-TERMINAL MUTATIONS PREVENT ACTIVATION BY RHODOPSIN- A NEW ASSAY USING RECOMBINANT PROTEINS EXPRESSED IN CULTURED-CELLS

We have measured the activation by recombinant rhodopsin of the alpha- subunit (alpha(t)) of retinal transducin (G(t), also recombinant) usin g a new assay. Cultured cells are transiently transfected with DNAs en coding opsin and the three subunits of C-t (alpha(t) beta(1) and gamma (1)). 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 GTP gamma S to protect alpha(t) fragments from proteolytic degrada tion. The activation of G(t) is also observed when all-trans-retinal i s added to microsomes under constant illumination. Activation depends on both opsin and retinal. Opsin mutants with known defects in activat ing G(t) show similar defects in this assay. alpha(t) mutations that m imic the corresponding mutations in the alpha-subunit of G(s) also pro duce qualitatively similar effects in this assay. As a first step in a strategy aimed at exploring the relationships between structure and f unction in the interactions of receptors with G proteins, we tested mu tant alpha(t) proteins with alanine substituted for each of the 10 ami no acids at the C-terminus, a region known to be crucial for interacti ons with rhodopsin. Alanine substitution at four positions moderately (K341) or severely (L344, G348, L349) impairs the susceptibility of al pha(t) to activation by rhodopsin. All four mutants retain their abili ty to be activated by AIF4(4)(-). Two other substitutions (N343 and F3 50) resulted in very mild defects, while substitutions at the remainin g four positions (E342, K345, D346 and C347) had no effect. In combina tion with previous observations, these results constrain models of the interaction of the C-terminus of alpha(t) with rhodopsin.