REGULATION OF THE RHODOPSIN TRANSDUCIN INTERACTION BY A HIGHLY CONSERVED CARBOXYLIC-ACID GROUP

Rhodopsin is a member of a family of G protein-coupled receptors which share structural and functional homologies. A tripeptide sequence (Gl u or Asp/Arg/Tyr) at the cytoplasmic border of the third transmembrane segment is conserved among most of these receptors. This region is in volved in G protein activation in rhodopsin as well as in other recept ors. The role of the conserved Glu-134 was studied by site-specific mu tagenesis of rhodopsin in combination with a real-time fluorescence as say of G protein (transducin) activation. Assay conditions were chosen under which the transducin activation rate was determined either by r hodopsin-transducin complex formation or by GTPgammaS-induced complex dissociation. Glu-134 was replaced by Gln in order to mimic the proton ated state of the carboxylic acid group. This mutation caused the pH d ependency of complex formation to extend to the alkaline range as comp ared with rhodopsin. Replacement of Glu-134 by Asp had an opposite but less pronounced effect on the pH dependency and lowered the overall e fficiency of transducin activation. The acidity constant (pK(a)) of th e residue at position 134 did not directly determine the pH sensitivit y of complex formation, indicating that other amino acid residues cont ribute to a titratable binding domain that includes Glu-134. In contra st, the pH sensitivity of GTPgammaS-induced complex dissociation was n ot changed by the mutations, although absolute rates were affected. Th e data suggest that the protonated state of Glu-134 favors binding of rhodopsin to transducin and that Glu-134 is not titratable in the rhod opsin-transducin complex.