Conditionally expressed G alpha 15 couples to endogenous receptors in GH3 cells.
The mammalian G proteins G15 and G16 couple a wide variety of receptors to phospholipase C (PLC) in co-transfected systems, and it has been suggested that they can be used as tools in agonist-screening systems. Using the reversed tetracycline-controlled transactivation system we generated rat pituitary GH3 cell clones that expressed Galphal5 and Galpha16 conditionally to study the coupling of endogenous receptors to both G proteins. In cells expressing moderate levels of Galpha15, activation of various endogenous receptors increased inositol phosphate production, whereas conditional expression of Galpha16 had no significant effect on agonist-dependent PLC activity. Activation of PLC through Galpha15 in response to carbachol did not increase cytosolic [Ca2+] ([Ca2+]i) but stimulated protein kinase C. While carbachol decreased the secretory activity in non-induced GH3 cells, it increased secretion in cells expressing Galpha15. Our data demonstrate that Galpha15 has a higher functional promiscuity than Galpha16 when studied in a system that preserves physiological G protein and receptor levels. In addition, Galpha15-mediated coupling of a receptor to PLC can change the cellular response to receptor agonists, indicating that downstream cellular functions can be used to detect receptor activation in screening systems employing a promiscuous G protein.
Offermanns
S
S
Pharmakologisches Institut, Universität Heidelberg, Germany. Stefan.Offermanns@urz.uni-heidelberg.de
Negulescu
P
P
Hu
Y H
YH
Simon
M I
MI
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism; D010957:Plasmids / Q000096:biosynthesis; D011388:Prolactin / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D007295:Inositol Phosphates; D011956:Receptors, Cell Surface; D011388:Prolactin; D011493:Protein Kinase C; C112631:G protein alpha 16; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D004318:Doxycycline