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
Op. Ernst et al., 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 Journal of biological chemistry, 270(18), 1995, pp. 10580-10586
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.