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

George K. Ehrlich, Matthew L. Andria, Xin Zheng, Brigitte Kieffer, Theresa L. Gioannini, Jacob M. Hiller, Jeremy E. Rosenkranz, Boris M. Veksler, R. Suzanne Zukin, Eric J. Simon

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Abstract

Previous work suggested that sulfhydryl groups and disulfide bridges have important functions in opioid binding to the δ opioid receptor. The question regarding which cysteines are essential for ligand binding was approached by replacement of cysteine residues in the cloned δ 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 δ agonist and antagonist binding activity. Immunofluorescence cytochemistry, performed with anti δ 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 δ 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.

Original languageEnglish (US)
Pages (from-to)269-277
Number of pages9
JournalCanadian Journal of Physiology and Pharmacology
Volume76
Issue number3
DOIs
StatePublished - 1998

Fingerprint

Opioid Receptors
Site-Directed Mutagenesis
Cysteine
Ligands
Disulfides
Sulfhydryl Reagents
Histocytochemistry
G-Protein-Coupled Receptors
Cricetulus
Cell Extracts
Alanine
Serine
Opioid Analgesics
Fluorescent Antibody Technique
Ovary
Binding Sites
Cell Membrane
Antibodies

Keywords

  • Cysteine
  • Opioid receptor
  • Site-directed mutagenesis
  • Sulfhydryl

ASJC Scopus subject areas

  • Physiology
  • Pharmacology

Cite this

Ehrlich, G. K., Andria, M. L., Zheng, X., Kieffer, B., Gioannini, T. L., Hiller, J. M., ... Simon, E. J. (1998). Functional significance of cysteine residues in the δ opioid receptor studied by site-directed mutagenesis. Canadian Journal of Physiology and Pharmacology, 76(3), 269-277. https://doi.org/10.1139/cjpp-76-3-269

Functional significance of cysteine residues in the δ opioid receptor studied by site-directed mutagenesis. / Ehrlich, George K.; Andria, Matthew L.; Zheng, Xin; Kieffer, Brigitte; Gioannini, Theresa L.; Hiller, Jacob M.; Rosenkranz, Jeremy E.; Veksler, Boris M.; Zukin, R. Suzanne; Simon, Eric J.

In: Canadian Journal of Physiology and Pharmacology, Vol. 76, No. 3, 1998, p. 269-277.

Research output: Contribution to journalArticle

Ehrlich, GK, Andria, ML, Zheng, X, Kieffer, B, Gioannini, TL, Hiller, JM, Rosenkranz, JE, Veksler, BM, Zukin, RS & Simon, EJ 1998, 'Functional significance of cysteine residues in the δ opioid receptor studied by site-directed mutagenesis', Canadian Journal of Physiology and Pharmacology, vol. 76, no. 3, pp. 269-277. https://doi.org/10.1139/cjpp-76-3-269
Ehrlich, George K. ; Andria, Matthew L. ; Zheng, Xin ; Kieffer, Brigitte ; Gioannini, Theresa L. ; Hiller, Jacob M. ; Rosenkranz, Jeremy E. ; Veksler, Boris M. ; Zukin, R. Suzanne ; Simon, Eric J. / Functional significance of cysteine residues in the δ opioid receptor studied by site-directed mutagenesis. In: Canadian Journal of Physiology and Pharmacology. 1998 ; Vol. 76, No. 3. pp. 269-277.
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