Functional Complementation and the Analysis of Opioid Receptor Homodimerization

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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 Gi1α 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 Giα immunoprecipitates from membranes of pertussis toxin-treated HEK293 cells. Agonist function was eliminated either by fusion of the receptors to Gi1αGly202Ala,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 Gi1αCys351Ile membrane anchored by linkage to DOP transmembrane domain 1. Inactive forms of all G protein α subunits can be produced by mutations equivalent to Gi1αGly202Ala. 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.