Antisense co-suppression of G(alpha)(q) and G(alpha)(11) demonstrates that both isoforms mediate M(3)-receptor-activated Ca(2+) signalling in intact epithelial cells.
We used replication-deficient adenoviruses overexpressing antisense against G(q) class alpha-subunits to determine the roles of G(q) and G(11) in mediating M(3)-receptor-coupled Ca(2+) mobilization in intact HT29 human colonic carcinoma epithelial cells. Western blot analysis and confocal microscopy showed that the viruses expressing antisense directed against the alpha-subunits of G(q) or G(11) produced isoform-specific reductions in the levels of these alpha-subunits. Fura-2 was used to measure changes in the Ca(2+) response following activation of the M(3) receptors by carbachol. The G(alpha)(q) antisense virus suppressed the peak Ca(2+) response by 70%, whereas the G(alpha)(11) antisense virus reduced it by 34%. We then used co-infection with both viruses to determine the effect of concomitant suppression of both G(alpha)(q) and G(alpha)(11). Overexpression of antisense to both alpha-subunits reduced by approximately 50% the levels of both G(alpha)(q) and G(alpha)(11). It also almost completely inhibited the Ca(2+) response to carbachol. These data show that both G(q) and G(11) are involved in mediating the action of the M(3) receptor on cytosolic Ca(2+) in HT29 cells. Furthermore, they suggest that the coupling of the M(3) receptor to these G proteins is specific, in that G(alpha)(q) cannot substitute for G(alpha)(11), and vice versa.
Department of Physiology, University of Sydney, Sydney, 2006 Australia.
M M
MM
Cummins
L M
LM
O'Mullane
J A
JA
Barden
D I
DI
Cook
P
P
Poronnik
D000256:Adenoviridae / Q000235:genetics; D020013:Calcium Signaling / Q000502:physiology*; D016373:DNA, Antisense; D004847:Epithelial Cells / Q000502:physiology*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D015967:Gene Expression Regulation, Viral; D019073:HT29 Cells; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000378:metabolism*
D016373:DNA, Antisense; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins