Recovery of Oligomers and Cooperativity When Monomers of the M2 Muscarinic Cholinergic Receptor Are Reconstituted into Phospholipid Vesicles

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• 作者：Amy W.-S. Ma ; Dar'ya S. Redka ; Luca F. Pisterzi ; St&eacute ; phane Angers ; James W. Wells
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：July 3, 2007
• 年：2007
• 卷：46
• 期：26
• 页码：7907 - 7927
• 全文大小：473K
• 年卷期：v.46,no.26(July 3, 2007)
• ISSN：1520-4995

文摘

FLAG- and HA-tagged M₂ muscarinic receptors from coinfected Sf9 cells have been purifiedin digitonin-cholate and reconstituted into phospholipid vesicles. The purified receptor was predominantlymonomeric: it showed no detectable coimmunoprecipitation; it migrated as a monomer duringelectrophoresis before or after cross-linking with bis(sulfosuccinimidyl)suberate; and it bound agonistsand antagonists in a manner indicative of identical and mutually independent sites. Receptor cross-linkedafter reconstitution or after reconstitution and subsequent solubilization in digitonin-cholate migratedalmost exclusively as a tetramer. The binding properties of the reconstituted receptor mimicked thosereported previously for cardiac muscarinic receptors. The apparent capacity for N-[³H]methylscopolamine(NMS) was only 60% of that for [³H]quinuclidinylbenzilate (QNB), yet binding at saturating concentrationsof [³H]QNB was inhibited fully and in a noncompetitive manner at comparatively low concentrations ofunlabeled NMS. Reconstitution of the receptor with a saturating quantity of functional G proteins led tothe appearance of three classes of sites for the agonist oxotremorine-M in assays with [³H]QNB; GMP-PNP caused an apparent interconversion from highest to lowest affinity and the concomitant emergenceof a fourth class of intermediate affinity. All of the data can be described quantitatively in terms ofcooperativity among four interacting sites, presumably within a tetramer; the effect of GMP-PNP can beaccommodated as a shift in the distribution of tetramers between two states that differ in their cooperativeproperties. Monomers of the M₂ receptor therefore can be assembled into tetramers with binding propertiesthat closely resemble those of the muscarinic receptor in myocardial preparations.