Use of replication-deficient adenoviruses to study signal transduction pathways. Muscarinic responses in HSG and HT29 epithelial cell lines are mediated by G protein betagamma-subunits.
HSG and HT29 cells express muscarinic receptors that increase intracellular free Ca2+ ([Ca2+]i) by activating phospholipase Cbeta. In the present study, we have used the measurement of [Ca2+]i with Fura-2 to show that these receptors are of the M3 sub-type and that the increase in [Ca2+]i triggered when they are activated is not sensitive to pertussis toxin. We have also used replication-deficient adenoviruses expressing wild-type and dominant-negative mutants of the alpha-subunits of the heterotrimeric G proteins, Gq and Gi2, to investigate the mechanisms by which these receptors control phospholipase Cbeta. We find that the Ca2+ response to 100 micromol/l carbachol is not affected by increased expression of the wild-type alpha-subunit of Gq, but is blocked by the dominant-negative mutant of Gq and by both the wild-type and the dominant-negative mutant alpha-subunits of Gi2. Expression of alpha-subunits of Gi2 presumably blocks the response to carbachol by scavenging free betagamma-subunits. We conclude that in HSG and HT29 cells, the Ca2+ response to M3 receptor activation is mediated by the betagamma- rather than the alpha-subunits of Gq.
Poronnik
P
P
Department of Physiology (F13), University of Sydney, NSW 2006 Australia.
O'Mullane
L M
LM
Conigrave
A D
AD
Cook
D I
DI
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics*; D020013:Calcium Signaling / Q000502:physiology; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D016257:Fura-2; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000502:physiology*; D019073:HT29 Cells; D006801:Humans; D018721:Muscarinic Agonists / Q000494:pharmacology; D009154:Mutation / Q000235:genetics / Q000502:physiology; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014779:Virus Replication / Q000235:genetics*
D018721:Muscarinic Agonists; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D000246:Adenosine Diphosphate Ribose; D002217:Carbachol; D019204:GTP-Binding Proteins; D016257:Fura-2