FUNCTIONAL-SIGNIFICANCE OF CYSTEINE RESIDUES IN THE DELTA-OPIOID RECEPTOR STUDIED BY SITE-DIRECTED MUTAGENESIS

Previous work suggested that sulfhydryl groups and disulfide bridges h ave important functions in opioid binding to the delta opioid receptor . The question regarding which cysteines are essential for ligand bind ing was approached by replacement of cysteine residues in the cloned d elta opioid receptor using site-directed mutagenesis. The wild-type an d mutant receptors were expressed stably in Chinese hamster ovary cell s. The two extracellular cysteine residues and the six located in tran smembrane domains were mutated to serine or alanine, one at a time. Re placement of either of the extracellular cysteines produced a receptor devoid of delta agonist and antagonist binding activity. Immunofluore scence cytochemistry, performed with anti delta opioid receptor antibo dies in washed cell monolayers in one of these mutants (Cys-Ser121), a nd immunoblots, performed on cell extracts, indicate that the receptor was expressed and seems to be associated with the cell membrane. The existence of an essential extracellular disulfide bridge, previously p ostulated by analogy to other G protein coupled receptors, is strongly supported by our results. Replacement of any one of the six transmemb rane cysteines was virtually without effect on the ability of the rece ptor to bind delta agonists and antagonists. Since there is strong evi dence that the transmembrane domains are involved in ligand binding, t hese results suggest that the cysteine residues, even those near or at the binding site, are not essential for receptor binding. Furthermore , these results support the idea that the striking effects of sulfhydr yl reagents on ligand binding of opioid receptors are likely to be due to steric hindrance by the large moieties transferred to the sulfhydr yl groups of cysteine residues by these reagents.