Abstract
Abstract: :
Purpose:To identify rhodopsin residues critical for transducin activation using a "gain-of-function" mutational approach. Rhodopsin regions targeted for mutagenesis included intracellular (IC) loop IC4 and non-helical portions of IC2 and IC3. Methods:Mutant opsins with residues 140-148 (IC2), 229-242 (IC3), or 310-320 (IC4) substituted by poly-Ala sequences of equivalent lengths served as templates for mutagenesis. Reverse substitutions of the Ala residues by rhodopsin residues have been generated in each of the templates. Additional mutant constructs were made with combinations of the reverse substitutions in different loops. Mutant opsins were expressed in COS7 cells. Plasma membrane preparations containing mutant opsins were regenerated with 11-cis-retinal, and the levels of recombinant pigments were determined from absorbance spectra. The ability of mutant rhodopsins to activate transducin was studied using GTPgS-binding assay. Results:The template constructs with poly-Ala substitutions in IC2-4 exhibited complete loss of the rhodopsin/transducin coupling. A near-full capacity for transducin activation was restored by the reverse substitutions of IC3 residues 229, 230, 240, and 242, and IC4 residues 310, 311, 313, and 317. Significant (~50%) restoration of the rhodopsin/transducin coupling was achieved with re-introduction of residues 140-141 into the IC2 Ala template. Analysis of rhodopsin mutants combining essential residues from the different IC loops reveal minimal structural requirements for transducin activation within the selected regions. Conclusions:Outside the helical bundle of rhodopsin, only a relatively small number of residues are important for the activation of transducin. These residues are adjacent to the transmembrane domains and are likely to directly contribute to the rhodopsin/transducin interface.
Keywords: photoreceptors • signal transduction • protein structure/function