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 (Glu or Asp/Arg/Tyr) at the cytoplasmic border of the third transmembrane segment is conserved among most of these receptors. This region is involved in G protein activation in rhodopsin as well as in other receptors. The role of the conserved Glu-134 was studied by site-specific mutagenesis of rhodopsin in combination with a real-time fluorescence assay of G protein (transducin) activation. Assay conditions were chosen under which the transducin activation rate was determined either by rhodopsin-transducin complex formation or by GTP gamma S-induced complex dissociation. Glu-134 was replaced by Gln in order to mimic the protonated state of the carboxylic acid group. This mutation caused the pH dependency of complex formation to extend to the alkaline range as compared with rhodopsin. Replacement of Glu-134 by Asp had an opposite but less pronounced effect on the pH dependency and lowered the overall efficiency of transducin activation. The acidity constant (pKa) of the residue at position 134 did not directly determine the pH sensitivity of complex formation, indicating that other amino acid residues contribute to a titratable binding domain that includes Glu-134. In contrast, the pH sensitivity of GTP gamma S-induced complex dissociation was not changed by the mutations, although absolute rates were affected. The data suggest that the protonated state of Glu-134 favors binding of rhodopsin to transducin and that Glu-134 is not titratable in the rhodopsin-transducin complex.
Fahmy
K
K
Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021.
Sakmar
T P
TP
D000595:Amino Acid Sequence; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism*; D017124:Conserved Sequence; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D015293:Transducin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D002264:Carboxylic Acids; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012243:Rhodopsin; D015293:Transducin