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 that activates the G protein transducin in response to light. Several site-directed rhodopsin mutants have been reported to be defective in transducin 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., König, B., Sakmar, T. P., Khorana, H. G., and Hofmann, K. P. (1990) Science 250, 123-125). The present study was carried 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 deletions from one cytoplasmic loop each: AB delta 70-71; CD delta 143-150; and EF delta 237-249. Two additional loop CD mutant genes were prepared: E134R/R135E had a reversal of a conserved charge pair, and CD r140-152 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 fluorescence assay of photoactivated rhodopsin (R*)-dependent guanosine 5'-O-(3-thiotriphosphate) uptake by transducin, 2) an assay of R*-dependent release of labeled GDP from the alpha-subunit of transducin holoenzyme (Gt alpha).GDP, and 3) a light-scattering assay of R*.Gt complex formation and dissociation. We show that the mutant pigments, which are able to bind transducin in a light-dependent manner but lack the ability to activate transducin, most likely form R*.Gt alpha beta gamma.GDP complexes that are impaired in GDP release.
Ernst
O P
OP
Howard Hughes Medical Institute, Laboratory of Molecular Biology and Biochemistry, Rockefeller University, New York, New York 10021, USA.
Hofmann
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KP
Sakmar
T P
TP
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008027:Light; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D015293:Transducin / Q000378:metabolism*
D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D012243:Rhodopsin; D015293:Transducin