Abstract
This paper reports a study of rhodopsin's structure and function using insertional mutagenesis with a flexible epitope. Sixteen rhodopsin derivatives were constructed, each of which carried a 12-amino acid epitope derived from the c-Myc protein flanked by penta-glycine linkers. For eight of the insertion mutants, the membrane sideness of the epitope insert was determined by immunostaining of intact or permeabilized cells. The results confirm the sidedness of each of the six helix connecting loops and the amino and carboxyl termini as postulated by the current seven-helix models of G-protein-coupled receptors and provide the first experimental evidence for the existence of the third extracellular loop. In general, inserts that were either closer to the amino terminus or on the extracellular face were more likely to disrupt folding and/or stability than were inserts near the carboxyl terminus or on the cytosolic face. Epitope insertion at positions 139 or 239, in the second and third cytosolic loops, respectively, failed to activate transducin, whereas an insertion at position 333 in the carboxyl-terminal tail was fully functional. The experimental approach described here should prove generally useful for elucidating structural and functional properties of both membrane and globular proteins.
Highlights
This paper reports a study of rhodopsin’s structure and chemical derivatization (Rohlich,1976;Martynov et a l . , and function using insertional mutagenesis with a flex-1983; Barclay andFindlay, 1984) shows that the amino termiible epitope
We describe the use of an insertional mutagenesis approach to analyze rhodopsin's stability, transmembrane topography, and ability t o activate transducin
The use of an epitope with glycine linkers increases the likelihood that insertion into the targetprotein will be tolerated and that theepitope will be accessibleto antibody binding
Summary
DNA and Cells-The mammalian expressionvector pCIS(Gormane.? al., 1990),bovine opsin cDNA (Nathans and Hogness, 1983), and the c-Myc epitope (MEQKLISEEDLNrecognized bymAb’Mycl-9ElO; Evan e.?al. (1985),KolodziejandYoung(1991),as modified by Wongand Cleveland (1990))have been described.For epitope insertion, a unique BamHI site was introduced by site-directed mutagenesis at various locations in the bovineopsin cDNAby replacing twocodons with GGATCC (encodingGly-Ser).The coding regionof eachBamHI-containing plasmid was cloned into a copy of the expressionvector that had not undergone the mutagenesis procedure, and wassequenced on one strand both to confirm the predicted mutations and to rule out spurious mutations. Membranes from transiently transfected cells expressing either wild type or mutant opsins were purified on a sucrose step gradient, incubated with 114s-retinal for several hours in the dark, sonicated for 10 s in 5 M urea to remove peripheral membrane proteins In one set of constructs the inserted sequence was flanked directlyby the BamHI cloning sites, and in the second set the inserted sequence was flanked on each side by five glycines The propertiesof these six constructs can be summarized as follows: 1)BPTI insertions failed to produce native opsin with or withougtlycine linkers; 2i)nfluenza hemaglutinin epitope insertions failed to produce native opsin without glycine linkers, but did produce native opsin with glycine linkers; and3) c-Myc epitope insertions produced native opsin with or without glycine linkers.
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