Heterooligomers of the muscarinic receptor and G proteins purified from porcine atria.
Muscarinic receptor extracted from porcine atria in digitonin-cholate copurified with Galpha(o), Galpha(i1-3), and caveolins. The presence of complexes was confirmed by coimmunoprecipitation of the receptor, alpha-subunits, and caveolins in various combinations. Homooligomers of alpha(i2) were detected on Western blots, and heterooligomers of alpha(i2) and alpha(o) were identified by coimmunoprecipitation; thus, a complex may contain at least two alpha-subunits. Other combinations of alpha-subunit were not detected. The ratio of total alpha-subunit to receptor was near 1, as measured by [(35)S]GTPgammaS and the antagonist [(3)H]quinuclidinylbenzilate, and the binding of [(35)S]GTPgammaS was manifestly biphasic. The ratio of alpha(o) to alpha(i1,2) 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 alpha-subunit, some of which are heteromeric. Each complex would appear to contain equal numbers of alpha-subunit and the receptor.
Ma
Amy W-S
AW
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ont., Canada M5S 3M2.
Pawagi
Asha B
AB
Wells
James W
JW
D000818:Animals; D022461:Caveolins / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000302:isolation & purification / Q000378:metabolism*; D006325:Heart Atria / Q000378:metabolism*; D018727:Muscarinic Antagonists / Q000494:pharmacology; D021122:Protein Subunits / Q000302:isolation & purification / Q000378:metabolism; D011813:Quinuclidinyl Benzilate / Q000494:pharmacology; D043585:Receptor, Muscarinic M2 / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013552:Swine / Q000378:metabolism*
D022461:Caveolins; D018727:Muscarinic Antagonists; D021122:Protein Subunits; D043585:Receptor, Muscarinic M2; D011813:Quinuclidinyl Benzilate; D019206:GTP-Binding Protein alpha Subunits, Gi-Go