Monomers and oligomers of the M2 muscarinic cholinergic receptor purified from Sf9 cells.
G protein-coupled receptors are known to form oligomers. To probe the nature of such aggregates, as well as the role and prevalence of monomers, epitope-tagged forms of the M(2) muscarinic receptor have been isolated as oligomers and monomers from Sf9 cells. Membranes from cells coexpressing the c-Myc- and FLAG-tagged receptor were solubilized in digitonin-cholate, and the receptor was purified by successive passage through DEAE-Sepharose, the affinity resin 3-(2'-aminobenzhydryloxy)tropane (ABT)-Sepharose, and hydroxyapatite. Coimmunoprecipitation of the two epitopes indicated the presence of oligomers at each stage of the purification up to but not including the fraction eluted specifically from ABT-Sepharose. The affinity-purified receptor therefore appeared to be monomeric. The failure to detect coimmunoprecipitation was not due to an ineffective antibody, nor did the conditions of purification appear to promote disaggregation. Receptor at all stages of purification bound N-[(3)H]methylscopolamine and [(3)H]quinuclidinylbenzilate with high affinity, but the capacity of receptors that were not retained on ABT-Sepharose was only 4% of that expected from densitometry of western blots probed with an anti-M(2) antibody. Similarly low activity was found with oligomers isolated by successive passage of coexpressed receptor on anti-c-Myc and anti-FLAG immunoaffinity columns. M(2) muscarinic receptors therefore appear to coexist as active monomers and largely or wholly inactive oligomers in solubilized extracts of Sf9 cells. A different pattern emerged when coinfected cells were treated with quinuclidinylbenzilate prior to solubilization, in that ABT-purified receptors from those cells exhibited coimmunoprecipitation. Treatment with the antagonist therefore led to oligomers in which at least some of the constituent sites were active and were retained by ABT-Sepharose.
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada M5S 2S2.
Paul S-H
PS
Park
James W
JW
Wells
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae; D002217:Carbachol / Q000737:chemistry; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D004072:Digitonin / Q000737:chemistry; D019281:Dimerization; D000939:Epitopes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D009842:Oligopeptides; D010455:Peptides / Q000378:metabolism; D011233:Precipitin Tests; D011438:Propylbenzilylcholine Mustard / Q000378:metabolism; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D011813:Quinuclidinyl Benzilate / Q000737:chemistry; D011869:Radioligand Assay; D043585:Receptor, Muscarinic M2 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D018411:Spodoptera / Q000737:chemistry / Q000378:metabolism / Q000821:virology
D000345:Affinity Labels; D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; D016271:Proto-Oncogene Proteins c-myc; D043585:Receptor, Muscarinic M2; D011438:Propylbenzilylcholine Mustard; D011813:Quinuclidinyl Benzilate; D002217:Carbachol; C085706:FLAG peptide; D004072:Digitonin