Abstract
In structure-function studies on bovine rhodopsin by in vitro site-specific mutagenesis, we have prepared three mutants in the cytoplasmic loop between the putative transmembrane helices E and F. In each mutant, charged amino acid residues were replaced by neutral residues: mutant 1, Glu239----Gln; mutant 2, Lys248----Leu; and mutant 3, Glu247----Gln, Lys248----Leu, and Glu249----Gln. The mutant rhodopsin genes were expressed in monkey kidney (COS-1) cells. After the addition of 11-cis-retinal to the cells, the rhodopsin mutants were purified by immunoaffinity adsorption. Each mutant gave a wild-type rhodopsin visible absorption spectrum. The mutants were assayed for their ability to stimulate the GTPase activity of transducin in a light-dependent manner. While mutants 1 and 3 showed wild-type activity, mutant 2 (Lys248----Leu) was inactive.
Highlights
In structure-function studies on bovine rhodopsin by in vitro site-specific mutagenesis, we have prepared three mutants in the cytoplasmic loop between the putative transmembrane helices E and F
Using the above expression system and in vitro site-specific mutagenesis of the synthetic rhodopsin gene, we are probing the sites of interaction between photolyzed rhodopsin andtransducin.Inthis communication, we report on the effects of amino acid substitutions in the cytoplasmic loop linking putative transmembranehelices E and F in rhodopsin [7] (Fig. 1).Existing data from proteolysis experiments of rhodopsin [8] and from deletion mutagenesis of the hamster /?-adrenergic receptor [9] suggested that this region was involved in Gprotein activation
A, rhodopsin purified from COS-1cells;B, mutant 1;C, mutant 2; D, mutant 3
Summary
In structure-function studies on bovine rhodopsin by in vitro site-specific mutagenesis, we have prepared three mutants in the cytoplasmic loop between the putative transmembrane helices E and F. The synthetic genewas expressed in COS-1 cells and the protein product, after in uiuo reconstitution with ll-cis-retinal, was purified, and characterized. Threemutant genes were constructed in which the following charged amino acids were replaced by neutral ones (Fig. 1): mutant 1, Glu’”+Gln; mutant 2, LysZ4’+Leu; and mutant3, a triple mutantG, Iu’~~+ Gln, Ly~’*~+Leu, anGdl ~ ~ * ~ + GAl nll .of the purified mutant proteins displayed wild-type rhodopsin absorption spectra.
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