Use of replication-deficient adenoviruses to study signal transduction pathways. Muscarinic responses in HSG and HT29 epithelial cell lines are mediated by G protein beta gamma-subunits

HSG and HT29 cells express muscarinic receptors that increase intracellular free Ca2+ ([Ca2+](i)) by activating phospholipase C beta. In the present s tudy, we have used the measurement of [Ca2+](i) with Fura-2 to show that th ese receptors are of the M-3 sub-type and that the increase in [Ca2+](i) tr iggered when they are activated is not sensitive to pertussis toxin. We hav e also used replication-deficient adenoviruses expressing wild-type and dom inant-negative mutants of the alpha-subunits of the heterotrimeric G protei ns, G(q) and G(i2), to investigate the mechanisms by which these receptors control phospholipase C beta. We find that the Ca2+ response to 100 mu mol/ l carbachol is not affected by increased expression of the wild-type alpha- subunit of G(q), but is blocked by the dominant-negative mutant of G(q) and by both the wild-type and the dominant-negative mutant alpha-subunits of G (i2). Expression of alpha-subunits of G(i2) presumably blocks the response to carbachol by scavenging free beta gamma-subunits. We conclude that in HS G and HT29 cells, the Ca2+ response to M-3 receptor activation is mediated by the beta gamma- rather than the alpha-subunits of G(q).