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

Abstract

 HSG and HT29 cells express muscarinic receptors that increase intracellular free Ca²⁺ ([Ca²⁺]_i) by activating phospholipase Cβ. 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 α-subunits of the heterotrimeric G proteins, G_q and G_i2, to investigate the mechanisms by which these receptors control phospholipase Cβ. We find that the Ca²⁺ response to 100 µmol/l carbachol is not affected by increased expression of the wild-type α-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 α-subunits of G_i2. Expression of α-subunits of G_i2 presumably blocks the response to carbachol by scavenging free βγ-subunits. We conclude that in HSG and HT29 cells, the Ca²⁺ response to M₃ receptor activation is mediated by the βγ- rather than the α-subunits of G_q.