Two-step mechanism of interaction of rhodopsin intermediates with the C-terminal region of the transducin alpha-subunit.
Rhodopsin is a prototypical G-protein-coupled receptor that contains 11-cis-retinal as a light-absorbing chromophore. Light causes conformational changes in the protein moiety through cis-trans isomerization of the chromophore, which leads to the formation of G-protein-interacting states. Our previous studies indicated that there are two intermediate states of rhodopsin, Meta Ib and Meta II, which interact differently with retinal G-protein transducin (Gt) [S. Tachibanaki, H. Imai, T. Mizukami, T. Okada, Y. Imamoto, T. Matsuda, Y. Fukada, A. Terakita, and Y. Shichida (1997) Biochemistry 36, 14173-14180]. Here we demonstrate that the interactions of Gt with these intermediates in the absence of GTPgammaS can be mimicked by the C-terminus 11-amino acid peptide (340-350) of the alpha-subunit of Gt (Gt(alpha)), suggesting that the C-terminal region of Gt(alpha) plays important roles in the interaction with rhodopsin intermediates. Replacement of either of the two leucine residues (Leu344 and Leu349) in the peptide with alanine caused the loss of the interaction with Meta II. However, the interaction with Meta Ib was abolished only when both residues were replaced. These results indicate that rearrangement of the C-terminal region of Gt(alpha) after the binding of a rhodopsin intermediate is necessary for the GDP-GTP exchange reaction on Gt(alpha).
Morizumi
Takefumi
T
Department of Biophysics, Graduate School of Science, Kyoto University, and Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Kyoto 606-8502.
Imai
Hiroo
H
Shichida
Yoshinori
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D007930:Leucine / Q000235:genetics; D008969:Molecular Sequence Data; D038761:Photobleaching; D012160:Retina / Q000737:chemistry; D012243:Rhodopsin / Q000031:analogs & derivatives* / Q000378:metabolism*; D013053:Spectrophotometry; D013997:Time Factors; D015293:Transducin / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism*
C013110:metarhodopsins; D012243:Rhodopsin; D015293:Transducin; D007930:Leucine