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.