[3H]propylbenzilylcholine mustard-labeling of muscarinic cholinergic receptors that selectively couple to phospholipase C or adenylate cyclase in two cultured cell lines.
Although both second messenger response systems are fully functional in both cell lines, activation of muscarinic cholinergic receptors only results in inhibition of adenylate cyclase in NG108-15 neuroblastoma X glioma cells and stimulation of phosphoinositide hydrolysis in 1321N1 human astrocytoma cells. Muscarinic receptors on both cell types were covalently labeled with [3H]propylbenzilylcholine mustard ([3H]PBCM), and the mobilities of the [3H]PBCM-labeled species of both cells were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 1321N1 and NG108-15 cells each primarily expressed a single [3H]PBCM-labeled species with an apparent size of approximately 92,000 and 66,000 Da, respectively. [3H]PBCM labeling was completely inhibited by 1 microM atropine or by down-regulation of muscarinic receptors by an overnight incubation with carbachol. The apparent size of the [3H]PBCM-labeled species of both cell lines was not altered by treatment with a series of protease inhibitors or by treatment with dithiothreitol and iodoacetamide. Since muscarinic receptors are glycoproteins, the contribution of carbohydrate groups to the difference in apparent size of the [3H]PBCM-labeled proteins was determined by treatment of [3H]PBCM-labeled membranes with endoglycosidase F, an enzyme that removes both complex and high mannose type N-linked carbohydrate chains. Endoglycosidase F treatment reduced the apparent size of the [3H]PBCM-labeled species in 1321N1 cells from 92,000 to approximately 77,000 Da and in NG108-15 cells from 66,000 to 45,000 Da. Neuraminidase produced no further reduction of the apparent size of the [3H]PBCM-labeled species from either cell after endoglycosidase F treatment, suggesting the absence of sialic acid containing O-linked carbohydrate chains on the muscarinic receptors of the two cell lines. The results suggest that different muscarinic receptor proteins may be responsible for the two different biochemical responses to muscarinic receptor activation.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D002794:Choline / Q000031:analogs & derivatives*; D006023:Glycoproteins / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D008024:Ligands; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D011438:Propylbenzilylcholine Mustard; D051381:Rats; D011976:Receptors, Muscarinic / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism*
D006023:Glycoproteins; D008024:Ligands; D008565:Membrane Proteins; D011976:Receptors, Muscarinic; D013438:Sulfhydryl Compounds; D011438:Propylbenzilylcholine Mustard; D010738:Type C Phospholipases; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D000262:Adenylyl Cyclases; D002794:Choline