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. |
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Authors: | R H Paulssen E J Paulssen K M Gautvik J O Gordeladze |
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Institution: | Institute of Medical Biochemistry, University of Oslo, Norway. |
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Abstract: | 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. |
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