The Gar , and Garll Proteins Couple the Thyrotropin - releasing Hormone Receptor to Phospholipase C in GH 3 Rat Pituitary Cells

Thyrotropin-releasing hormone stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP4 in GH3 cell membranes. The stimulation of the phosphoinositide phospholipase C (PI/PLC) activity can be blocked by incubation of GH3 membranes with polyclonal antibodies directed against a peptide derived from the C-terminal region of Gaq and G a l l . Antibodies directed against the C-terminal region of other Ga-subunits had no detectable effect. The inhibition was specific since addition of the peptide that was used to prepare the antibody completely reversed the inhibition. Further evidence for the coupling of the TRH receptor to Ga, or Gall comes from a reconstitution experiment in which human embryonic kidney cells were transiently transfected with cDNAs corresponding to the TRH receptor, Ga, or Gall. The PIP2 hydrolysis detected with membranes from cells that over-expressed the TRH receptor alone was low, however, co-expression with the Ga, or Gall subunits produced a synergistic stimulation of PI-PLC activity. In contrast, co-expression of these a-subunits with the M2 muscarinic acetylcholine receptor induced a weak stimulation of PIPz hydrolysis. The results presented here suggest that the TRH-dependent stimulation of PI-PLC in GH3 cells is mediated through the G-protein a-subunits, Ga, and/or Gall.