Rhodopsin-interacting surface of the transducin gamma subunit.
The visual signaling pathway is initiated by photoactivation of the GPCR rhodopsin, which activates nucleotide exchange on the heterotrimeric G-protein transducin (Gt). Domains on both Gtalpha and Gtbetagamma subunits participate in coupling to rhodopsin. Previously, we have shown by high-resolution NMR that the farnesylated C-terminal peptide of Gtgamma(60-71), DKNPFKELKGGC, assumes an amphipathic helical conformation during interaction with metarhodopsin II [Kisselev, O. G., and Downs, M. A. (2003) Structure 11, 367-373]. This conformation was docked to the structure of holo-Gt to create a model of rhodopsin-Gt interaction. Here we test this model by mutational analysis of Gt. To evaluate the contribution of specific amino acids of the Gtgamma C-terminal region involved in binding and GTP-dependent release of transducin from native rhodopsin membranes, we have systematically substituted each of the amino acids in the C-terminal region of Gtgamma for alanine. The mutants were co-expressed with six-histidine-tagged Gtbeta subunits in Sf9 insect cells. The Gtbeta-6-His-gamma mutant proteins were purified and assayed in the presence of Gtalpha for the GTP-dependent interactions with light-activated rhodopsin. Several of the alanine mutants, N62A, P63A, and F64A, exhibited significant functional defects at the level of R*-Gt complex formation. These data show that the conserved N-terminal end of the helical domain in the Gtgamma(60-71) region has the most significant effect on rhodopsin-Gt interactions, which places important constraints on the model of the rhodopsin-Gt complex.
Kisselev
Oleg G
OG
Department of Ophthalmology, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA. kisselev@slu.edu
Downs
Maureen A
MA
D000409:Alanine / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D012243:Rhodopsin / Q000378:metabolism*; D015293:Transducin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D012243:Rhodopsin; D015293:Transducin; D000409:Alanine