Abstract

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_i1α was linked to the C-terminal tails of the δ opioid peptide (DOP), κ opioid peptide, and μ opioid peptide receptors. Each was functional as measured by agonist stimulation of guanosine 5′-([γ-³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding in G_iα immunoprecipitates from membranes of pertussis toxin-treated HEK293 cells. Agonist function was eliminated either by fusion of the receptors to G_i1αGly²⁰²Ala,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]GTPγS for each receptor. At equimolar amounts, reconstitution of DOP receptor function was more extensive than κ or μ opioid receptor. Reconstitution of DOP function required two intact receptors and was not achieved by provision of extra G_i1αCys³⁵¹Ile membrane anchored by linkage to DOP transmembrane domain 1. Inactive forms of all G protein α subunits can be produced by mutations equivalent to G_i1αGly²⁰²Ala. 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.