Kinetics and reversibility of thyrotropin-releasing hormone-stimulated guanine nucleotide exchange in membranes from GH4C1 cells.

To evaluate the role of thyrotropin-releasing hormone (TRH)-stimulated guanine nucleotide exchange in the biphasic cellular responses to TRH, we have examined the kinetics, reversibility, and inhibition by QC120 (an antiserum recognizing the carboxyl terminus of alpha q/11) of TRH-stimulated guanosine-59-(alpha-[35S] thio)triphosphate ([35S]GTP alpha S) binding in membranes from GH4C1 cells. Enhanced binding of [35S]GTP alpha S stimulated by TRH was dose dependent and readily detectable within 8 sec of TRH treatment. Binding measured within the first 20 sec was largely inhibited by QC120, whereas additional binding that accumulated during incubations of 3-6 min was not inhibited by even high concentrations of the antiserum. TRH-stimulated binding was reversible, in that, after membranes were incubated with TRH and [35S]GTP alpha S, subsequent addition of excess GTP caused exchange of 70-100% of the prebound radioligand. Exchange of TRH-stimulated [35S]GTP alpha S binding occurred in fast and slow phases, with half-times of