Purification and Functional Reconstitution of Monomeric μ-Opioid Receptors ALLOSTERIC MODULATION OF AGONIST BINDING BY G

• Published in: The Journal of biological chemistry Vol. 284; no. 39; pp. 26732 - 26741
• Main Authors: Kuszak, Adam J., Pitchiaya, Sethuramasundaram, Anand, Jessica P., Mosberg, Henry I., Walter, Nils G., Sunahara, Roger K.
• Format: Journal Article
• Language: English
• Published: 9650 Rockville Pike, Bethesda, MD 20814, U.S.A American Society for Biochemistry and Molecular Biology 25.09.2009
• Subjects: Mechanisms of Signal Transduction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.026922

Despite extensive characterization of the μ-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 μ-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′-(γ-thio)triphosphate binding to Gα 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 μ-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.