Heterooligomers of the muscarinic receptor and G proteins purified from porcine atria

Abstract

Muscarinic receptor extracted from porcine atria in digitonin–cholate copurified with Gαo, Gαi1–3, and caveolins. The presence of complexes was confirmed by coimmunoprecipitation of the receptor, α-subunits, and caveolins in various combinations. Homooligomers of αi2 were detected on Western blots, and heterooligomers of αi2 and αo were identified by coimmunoprecipitation; thus, a complex may contain at least two α-subunits. Other combinations of α-subunit were not detected. The ratio of total α-subunit to receptor was near 1, as measured by [35S]GTPγS and the antagonist [3H]quinuclidinylbenzilate, and the binding of [35S]GTPγS was manifestly biphasic. The ratio of αo to αi1,2 also was near 1, as determined from the intensity of Western blots. Cardiac muscarinic receptors therefore can be purified as a mixture of complexes that contain caveolins and oligomers of α-subunit, some of which are heteromeric. Each complex would appear to contain equal numbers of α-subunit and the receptor.