RHODOPSIN ACTIVATION BLOCKED BY METAL-ION-BINDING SITES LINKING TRANSMEMBRANE HELICE-C AND HELICE-F

A LARGE superfamily of receptors containing seven transmembrane (TRI) helices transmits hormonal and sensory signals across the plasma membr ane to heterotrimeric G proteins at the cytoplasmic face of the membra ne. To investigate how G-protein-coupled receptors work at the molecul ar level, we have engineered metal-ion-binding sites between TM helice s to restrain activation-induced conformational change in specific loc ations. In rhodopsin, the photoreceptor of retinal rod cells, we subst ituted histidine residues for natural amino acids at the cytoplasmic e nds 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 helice s C and F are in close proximity and suggest that movements of these h elices relative to one another are required for transducin activation. Thus a change in the orientations of TRI 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 rece ptors.