Nature of the oligomers formed by muscarinic m2 acetylcholine receptors in Sf9 cells

Abstract

Wild-type, FLAG-tagged, and c-myc-tagged muscarinic m2 receptors extracted in digitonin–cholate from singly and co-infected Sf9 ( Spodoptera frugiperda) cells were indistinguishable in their binding of [ 3H]quinuclidinylbenzilate, either before or after purification. The FLAG epitope was found to coimmunoprecipitate with the c-myc epitope when co-infected cells were solubilised in digitonin–cholate, n-dodecyl-β- d-maltoside or Lubrol-PX. The degree of coprecipitation in digitonin–cholate was unaffected by preincubation of the extract for up to 60 min at 30°C, with or without muscarinic receptor ligands; no coimmunoprecipitation occurred in mixed extracts from singly infected cells. As measured by [ 3H]quinuclidinylbenzilate, the efficiency of immunoprecipitation from co-infected cells was 87% of that from singly infected cells. The amount of receptor immunoprecipitated from the latter, as determined by densitometry, was 2.3-fold that expected from the loss of binding from the extract. The data suggest that at least some of the receptors were trimeric or larger and that oligomers neither formed nor dissociated under the conditions of the experiments. Also, some receptors appear to be non-functional or latent in digitonin-solubilised extracts.