title: Purification and functional reconstitution of monomeric mu-opioid receptors: allosteric modulation of agonist binding by Gi2.
abstract: Despite extensive characterization of the mu-opioid receptor (MOR), the biochemical properties of the isolated receptor remain unclear. In light of recent reports, we proposed that the monomeric form of MOR can activate G proteins and be subject to allosteric regulation. A mu-opioid receptor fused to yellow fluorescent protein (YMOR) was constructed and expressed in insect cells. YMOR binds ligands with high affinity, displays agonist-stimulated [(35)S]guanosine 5'-(gamma-thio)triphosphate binding to Galpha(i), and is allosterically regulated by coupled G(i) protein heterotrimer both in insect cell membranes and as purified protein reconstituted into a phospholipid bilayer in the form of high density lipoprotein particles. Single-particle imaging of fluorescently labeled receptor indicates that the reconstituted YMOR is monomeric. Moreover, single-molecule imaging of a Cy3-labeled agonist, [Lys(7), Cys(8)]dermorphin, illustrates a novel method for studying G protein-coupled receptor-ligand binding and suggests that one molecule of agonist binds per monomeric YMOR. Together these data support the notion that oligomerization of the mu-opioid receptor is not required for agonist and antagonist binding and that the monomeric receptor is the minimal functional unit in regard to G protein activation and strong allosteric regulation of agonist binding by G proteins.
authors: Departments of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109, USA. .
authors: Adam J
authors: AJ
authors: Kuszak
authors: Sethuramasundaram
authors: S
authors: Pitchiaya
authors: Jessica P
authors: JP
authors: Anand
authors: Henry I
authors: HI
authors: Mosberg
authors: Nils G
authors: NG
authors: Walter
authors: Roger K
authors: RK
authors: Sunahara
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000701:Analgesics, Opioid / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002460:Cell Line; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000378:metabolism / Q000494:pharmacology; D051960:GTP-Binding Protein alpha Subunit, Gi2 / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D008075:Lipoproteins, HDL / Q000737:chemistry / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D018847:Opioid Peptides / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D017450:Receptors, Opioid, mu / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
chemical_list: D000701:Analgesics, Opioid; D008075:Lipoproteins, HDL; D008164:Luminescent Proteins; D018847:Opioid Peptides; D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; C030058:dermorphin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D051960:GTP-Binding Protein alpha Subunit, Gi2