Further evidence that muscarinic cholinergic receptors of 1321N1 astrocytoma cells couple to a guanine nucleotide regulatory protein that is not Ni.
Inhibitory coupling of receptors to adenylate cyclase previously has been shown to be relatively sensitive to inactivation by alkylation with N-ethylmaleimide (NEM). Modification of the inhibitory guanine nucleotide regulatory protein, Ni, has been proposed to be responsible for this effect. The effects of NEM on GTP-sensitive binding of carbachol to muscarinic cholinergic receptors has been compared in a cell line (1321N1 human astrocytoma cells) in which these receptors stimulate phosphoinositide breakdown and in a cell line (NG108-15 neuroblastoma X glioma cells) in which activation of these receptors results in inhibition of adenylate cyclase. Pretreatment of membrane preparations from 1321N1 cells with NEM resulted in a concentration-dependent decrease in the extent of pertussis toxin-catalysed [32P]ADP-ribosylation of a 41 000 Da protein previously proposed to be the alpha subunit of Ni. Under conditions where 32P-labelling of Ni in 1321N1 membranes was reduced by NEM by 90%, no effect was observed on the extent of guanine nucleotide-sensitive high-affinity binding of carbachol to muscarinic cholinergic receptors. In contrast, treatment of NG108-15 membranes with NEM under the same conditions resulted in complete loss of high-affinity guanine nucleotide sensitive binding of carbachol. These results illustrate another difference between the muscarinic receptor population of these two cell lines, and support the previous proposal that muscarinic receptors of 1321N1 cells couple to a guanine nucleotide regulatory protein that is not Ni.
D000244:Adenosine Diphosphate / Q000378:metabolism; D037361:Adenylate Cyclase Toxin; D001254:Astrocytoma / Q000378:metabolism*; D001427:Bacterial Toxins / Q000494:pharmacology; D002217:Carbachol / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D037342:Pertussis Toxin; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism*; D010566:Virulence Factors, Bordetella
D037361:Adenylate Cyclase Toxin; D001427:Bacterial Toxins; D008565:Membrane Proteins; D011976:Receptors, Muscarinic; D010566:Virulence Factors, Bordetella; D000244:Adenosine Diphosphate; D002217:Carbachol; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D005033:Ethylmaleimide