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-5'-(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 < 5 sec and 187 sec, respectively. Addition of QC120 before the GTP chase inhibited the fast phase of exchange, whereas reduction of the TRH concentration in the preincubation selectively reduced the magnitude of the slow phase. Neither phase of exchange was affected by prior treatment of cells with pertussis toxin. Our observations indicate that Gq/11 is rapidly activated by the TRH receptor and that a second, unidentified, G protein is slowly activated by the TRH receptor.
Brady
K D
KD
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, Massachusetts.
Han
B
B
Tashjian
A H
AH
D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D007106:Immune Sera; D007700:Kinetics; D008969:Molecular Sequence Data; D037342:Pertussis Toxin; D013973:Thyrotropin-Releasing Hormone / Q000502:physiology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007106:Immune Sera; D010566:Virulence Factors, Bordetella; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D013973:Thyrotropin-Releasing Hormone; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins