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Functional significance of cysteine residues in the delta opioid receptor studied by site-directed mutagenesis.

Abstract：

:Previous work suggested that sulfhydryl groups and disulfide bridges have important functions in opioid binding to the delta opioid receptor. The question regarding which cysteines are essential for ligand binding was approached by replacement of cysteine residues in the cloned delta opioid receptor using site-directed mutagenesis. The wild-type and mutant receptors were expressed stably in Chinese hamster ovary cells. The two extracellular cysteine residues and the six located in transmembrane domains were mutated to serine or alanine, one at a time. Replacement of either of the extracellular cysteines produced a receptor devoid of delta agonist and antagonist binding activity. Immunofluorescence cytochemistry, performed with anti delta opioid receptor antibodies in washed cell monolayers in one of these mutants (Cys-Ser121), and 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 postulated by analogy to other G protein coupled receptors, is strongly supported by our results. Replacement of any one of the six transmembrane cysteines was virtually without effect on the ability of the receptor to bind delta agonists and antagonists. Since there is strong evidence that the transmembrane domains are involved in ligand binding, these 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 sulfhydryl reagents on ligand binding of opioid receptors are likely to be due to steric hindrance by the large moieties transferred to the sulfhydryl groups of cysteine residues by these reagents.

journal_name

Can J Physiol Pharmacol

journal_title

Canadian journal of physiology and pharmacology

authors

Ehrlich GK,Andria ML,Zheng X,Kieffer B,Gioannini TL,Hiller JM,Rosenkranz JE,Veksler BM,Zukin RS,Simon EJ

subject

Has Abstract

pub_date

1998-03-01 00:00:00

pages

269-77

issue

eissn

0008-4212

issn

1205-7541

journal_volume

pub_type

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Functional significance of cysteine residues in the delta opioid receptor studied by site-directed mutagenesis.