Use of replication deficient adenoviruses to investigate the role of G proteins in Ca2+ signalling in epithelial cells.
Here we report on the feasibility of using replication deficient adenoviruses to modify signal transduction systems in epithelia. We constructed two viruses, one expressing a dominant negative mutant of the alpha-subunit of Gq (Ad-EF1-dnG alpha q) and the other expressing the wild-type alpha-subunit of Gq (Ad-EF1-wtG alpha q). We used an adenovirus expressing green fluorescent protein (Ad-EF1-GFP20) to show that infection of cultured cells with an adenovirus results in at least 95% expression of the transgene in both HSG and HT29 cells. We also used an adenovirus that expresses no transgene (Ad-MX17) to demonstrate that adenoviral infection itself does not affect the resting concentration of cytosolic Ca2+ ([Ca2+]i) or the carbachol responses in these cells. We further show that Ad-EF1-dnG alpha q inhibits the increase in [Ca2+]i produced by muscarinic receptor activation in both the cell lines we studied. This inhibitory effect is not shared by Ad-EF1-wtG alpha q, which indicates that in both HSG and HT29 cells, the increase in [Ca2+]i produced by muscarinic receptor activation is largely mediated by activation of Gq. Neither virus affected the resting level of [Ca2+]i in these cells. Our findings confirm the feasibility of using replication deficient adenoviruses expressing dominant negative mutants to investigate the role of G proteins in signal transduction systems.
Poronnik
P
P
Department of Physiology, University of Sydney, New South Wales, Australia.
O'Mullane
L M
LM
Harding
E A
EA
Greger
R
R
Cook
D I
DI
D000256:Adenoviridae / Q000235:genetics*; D020013:Calcium Signaling; D002217:Carbachol / Q000494:pharmacology; D002277:Carcinoma / Q000821:virology; D003110:Colonic Neoplasms / Q000821:virology; D004847:Epithelial Cells / Q000378:metabolism* / Q000821:virology; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018721:Muscarinic Agonists; D009154:Mutation; D011976:Receptors, Muscarinic / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013363:Submandibular Gland / Q000166:cytology / Q000821:virology; D014407:Tumor Cells, Cultured; D014779:Virus Replication
D008164:Luminescent Proteins; D018721:Muscarinic Agonists; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D002217:Carbachol; D019204:GTP-Binding Proteins