The thyroliberin receptor interacts directly with a stimulatory guanine-nucleotide-binding protein in the activation of adenylyl cyclase in GH3 rat pituitary tumour cells. Evidence obtained by the use of antisense RNA inhibition and immunoblocking of the stimulatory guanine-nucleotide-binding protein.
The thyroliberin receptor in GH3 pituitary tumour cells is known to couple to phospholipase C via a guanine-nucleotide-binding protein (G protein). Thyroliberin is postulated also to activate adenylyl cyclase, via the stimulatory G protein (Gs). In order to study this coupling, we constructed an antisense RNA expression vector that contained part of the Gs alpha-subunit cDNA clone (Gs alpha) in an inverted orientation relative to the mouse metallothionein promoter. The cDNA fragment included part of the coding region and all of the 3' non-translated region. Transient expression of Gs alpha antisense RNA in GH3 cells resulted in the specific decrease of Gs alpha mRNA levels, followed by decreased Gs alpha protein levels. Thyroliberin-elicited adenylyl cyclase activation in membrane preparations showed a reduction of up to 85%, whereas phospholipase C stimulation remained unaffected. Activation of adenylyl cyclase by vasoactive intestinal peptide was reduced by 30-40%. Investigation of the effects of thyroliberin and vasoactive intestinal peptide on adenylyl cyclase in GH3 cell membranes pretreated with antisera against Gs alpha and Gi-1 alpha/Gi-2 alpha support the results obtained by the use of the antisense technique. We conclude that thyroliberin has a bifunctional effect on GH3 cells, in activating adenylyl cyclase via Gs or a Gs-like protein in addition to the coupling to phospholipase C.
Paulssen
R H
RH
Institute of Medical Biochemistry, University of Oslo, Norway.
Paulssen
E J
EJ
Gautvik
K M
KM
Gordeladze
J O
JO
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D015870:Gene Expression; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007106:Immune Sera; D010911:Pituitary Neoplasms / Q000201:enzymology*; D010957:Plasmids; D016372:RNA, Antisense / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017981:Receptors, Neurotransmitter / Q000502:physiology*; D018025:Receptors, Thyrotropin-Releasing Hormone; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology
D007106:Immune Sera; D016372:RNA, Antisense; D012333:RNA, Messenger; D017981:Receptors, Neurotransmitter; D018025:Receptors, Thyrotropin-Releasing Hormone; D006165:Guanylyl Imidodiphosphate; D014660:Vasoactive Intestinal Peptide; D013973:Thyrotropin-Releasing Hormone; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases