Mutation of human μ opioid receptor extracellular "disulfide cysteine'' residues alters ligand binding but does not prevent receptor targeting to the cell plasma membrane

The FL opioid receptor, a primary site of action in the brain for opioid neuropeptides and opiate drugs of abuse, is a member of the seven transmembrane, G protein-coupled receptor (GPCR) superfamily. Two cysteine residues, one in each of the first two of three extracellular loops (ECLs), are highly conserved among GPCRs, and there is direct or circumstantial evidence that the residues form a disulfide bond in many of these receptors. Such a bond would dramatically govern the topology of the ECLs, and possibly affect the position of the membrane-spanning domains. Recent findings from several laboratories indicate the importance of the ECLs for opioid ligand selectivity. These conserved cysteine residues in the mu opioid receptor were studied using site-directed mutagenesis. Little or no specific binding of radiolabled opiate alkaloid or opioid peptide agonists or antagonists was observed for receptors mutated at either "disulfide cysteine" residue. Each mutant mu opioid receptor was expressed in both transiently- and stably-transfected cells, in some cases at levels comparable to the wild type receptor. The two point mutants possessing serine-for-cysteine substitutions were also observed to successfully reach the cell plasma membrane, as evidenced by electron microscopy. Consistent with related work with other GPCRs, the mu opioid receptor apparently also employs the extracellular disulfide bond. This information now permits accurate molecular modeling of extracellular aspects of the receptor, including plausible scenarios of mu receptor docking of opioid Ligands known to require specific extracellular loop features for high affinity binding. (C) 1999 Elsevier Science B.V. All rights reserved.