Nature 383, 347 - 350 (26 September 1996); doi:10.1038/383347a0

Rhodopsin activation blocked by metal-ion-binding sites linking transmembrane helices C and F

Søren P. Sheikh^*, Tatyana A. Zvyaga^†, Olivier Lichtarge^*, Thomas P. Sakmar^† & Henry R. Bourne^*

^* Department of Cellular and Molecular Biology and the Cardiovascular Research Institute, University of California, San Francisco, California 94143, USA
^† Howard Hughes Medical Institute, Laboratory of Molecular Biology and Biochemistry, Rockefeller University, New York, New York 10021, USA

A LARGE superfamily of receptors containing seven transmembrane (TM) helices transmits hormonal and sensory signals across the plasma membrane to heterotrimeric G proteins at the cytoplasmic face of the membrane. To investigate how G-protein-coupled receptors work at the molecular level, we have engineered metal-ion-binding sites between TM helices to restrain activation-induced conformational change in specific locations. In rhodopsin, the photoreceptor of retinal rod cells, we substituted histidine residues for natural amino acids at the cytoplasmic ends of the TM helices C and F. The resulting mutant proteins were able to activate the visual G protein transducin in the absence but not in the presence of metal ions. These results indicate that the TM helices C and F are in close proximity and suggest that movements of these helices relative to one another are required for transducin activation. Thus a change in the orientations of TM helices C and F is likely to be a key element in the mechanism for coupling binding of ligands (or isomerization of retinal) to the activation of G-protein-coupled receptors.

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