CHARACTERIZATION OF RHODOPSIN MUTANTS THAT BIND TRANSDUCIN BUT FAIL TO INDUCE GTP NUCLEOTIDE UPTAKE - CLASSIFICATION OF MUTANT PIGMENTS BY FLUORESCENCE, NUCLEOTIDE RELEASE, AND FLASH-INDUCED LIGHT-SCATTERING ASSAYS

The photoreceptor rhodopsin is a seven-transmembrane helix receptor th at activates the G protein transducin in response to light. Several si te-directed rhodopsin mutants have been reported to be defective in tr ansducin activation. Two of these mutants bound transducin in response to light, but failed to release the bound transducin in the presence of GTP (Franke, R. R., Konig, B., Sakmar, T. P., Khorana, H. G., and H ofmann, K. P. (1990) Science 250, 123-125). The present study was carr ied out to determine the nucleotide-binding state of transducin as it interacts with rhodopsin mutants. Five mutant bovine opsin genes were prepared by site-specific mutagenesis. Three mutant genes had deletion s from one cytoplasmic loop each: AB Delta 70-71; CD Delta 143-150; an d EF Delta 237-249. Two additional loop CD mutant genes were prepared: E134R/R135E had a reversal of a conserved charge pair, and CD r140-15 2 had a 13-amino acid sequence replaced by a sequence derived from the amino terminal tail. Three types of assays were carried out: 1) a flu orescence assay of photoactivated rhodopsin (R) dependent guanosine 5 '-O-(3-thiotriphosphate) uptake by transducin, 2) an assay of R-depen dent release of labeled GDP from the alpha-subunit of transducin holoe nzyme (G(t alpha)). GDP, and 3) a light-scattering assay of R G(t) co mplex formation and dissociation. We show that the mutant pigments, wh ich are able to bind transducin in a light-dependent manner but lack t he ability to activate transducin, most likely form R. G(t alpha beta gamma). GDP complexes that are impaired in GDP release.