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Functional Complementation and the Analysis of Opioid Receptor Homodimerization

Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom

Complementation of function after coexpression of pairs of nonfunctional G protein-coupled receptors that contain distinct inactivating mutations supports the hypothesis that such receptors exist as dimers. Chimeras between members of the metabotropic glutamate receptor-like family have been particularly useful because the N-terminal ligand binding and heptahelical transmembrane elements can be considered distinct domains. To examine the utility of a related approach for opioid receptors, fusion proteins were generated in which a pertussis toxin-resistant (Cys³⁵¹Ile) variant of the G protein G_i1 was linked to the C-terminal tails of the opioid peptide (DOP), opioid peptide, and µ opioid peptide receptors. Each was functional as measured by agonist stimulation of guanosine 5'-([-³⁵S]thio)triphosphate ([³⁵S]GTPS) binding in G_i immunoprecipitates from membranes of pertussis toxin-treated HEK293 cells. Agonist function was eliminated either by fusion of the receptors to G_i1Gly²⁰²Ala,Cys³⁵¹Ile or mutation of a pair of conserved Val residues in intracellular loop 2 of each receptor. Coexpression, but not simple mixing, of the two inactive fusion proteins reconstituted agonist-loading of [³⁵S]GTPS for each receptor. At equimolar amounts, reconstitution of DOP receptor function was more extensive than or µ opioid receptor. Reconstitution of DOP function required two intact receptors and was not achieved by provision of extra G_i1Cys³⁵¹Ile membrane anchored by linkage to DOP transmembrane domain 1. Inactive forms of all G protein subunits can be produced by mutations equivalent to G_i1Gly²⁰²Ala. Because the amino acids modified in the opioid receptors are highly conserved in most rhodopsin-like receptors, this approach should be widely applicable to study the existence and molecular basis of receptor dimerization.

Address correspondence to: Graeme Milligan, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK. E-mail: g.milligan{at}bio.gla.ac.uk

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