A constitutively active mutant thyrotropin-releasing hormone receptor is chronically down-regulated in pituitary cells: evidence using chlordiazepoxide as a negative antagonist

Mol Endocrinol. 1995 Nov;9(11):1455-60. doi: 10.1210/mend.9.11.8584022.


A carboxyl-terminus truncated mutant of the guanine nucleotide-binding (G) protein-coupled TRH receptor (TRH-R) was previously shown to exhibit constitutive, i.e. TRH-independent, activity (C335Stop TRH-R). Chlordiazepoxide (CDE), a known competitive inhibitor of TRH binding to wild-type (WT) TRH-Rs, is shown to compete for binding to C335Stop TRH-Rs also. More importantly, CDE is shown to be a negative antagonist of C335Stop TRH-Rs. CDE rapidly caused the basal rate of inositol phosphate second messenger (IP) formation to decrease in AtT-20 pituitary cells stably expressing C335Stop TRH-Rs (AtT-C335Stop cells), but not in cells expressing WT TRH-Rs (AtT-WT cells). Similar observations were made in HeLa cells transiently expressing C335Stop or WT TRH-Rs. CDE inhibition of IP formation was shown to be specific for TRH-Rs using GH4C1 cells expressing both TRH-Rs and receptors for bombesin. In these cells, CDE inhibited TRH-stimulated IP formation, but had no effect on bombesin-stimulated IP formation. The effects of chronic administration of CDE were studied. Preincubation of AtT-C335Stop cells, but not AtT-WT cells, with CDE for several hours caused an increase in cell surface receptor number (up-regulation) that led to increased TRH stimulation of inositol phosphate formation and elevation of intracellular free Ca2+. Preincubation with CDE did not affect methyl-TRH binding affinity or TRH potency in cells expressing AtT-C335Stop or in AtT-WT cells. We conclude that CDE is a negative antagonist of C335Stop TRH-Rs and that constitutively active C335Stop TRH-Rs are down-regulated in AtT-20 pituitary cells in the absence of agonist.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Binding, Competitive
  • Chlordiazepoxide / metabolism
  • Chlordiazepoxide / pharmacology*
  • Down-Regulation*
  • HeLa Cells / metabolism
  • Humans
  • Inositol Phosphates / metabolism
  • Mice
  • Pituitary Gland, Anterior / metabolism*
  • Pituitary Neoplasms / pathology
  • Protein Conformation
  • Receptors, Bombesin / metabolism
  • Receptors, Thyrotropin-Releasing Hormone / biosynthesis
  • Receptors, Thyrotropin-Releasing Hormone / chemistry
  • Receptors, Thyrotropin-Releasing Hormone / drug effects
  • Receptors, Thyrotropin-Releasing Hormone / genetics*
  • Receptors, Thyrotropin-Releasing Hormone / metabolism
  • Second Messenger Systems / drug effects
  • Thyrotropin-Releasing Hormone / antagonists & inhibitors*
  • Thyrotropin-Releasing Hormone / metabolism
  • Tumor Cells, Cultured
  • Up-Regulation / drug effects


  • Inositol Phosphates
  • Receptors, Bombesin
  • Receptors, Thyrotropin-Releasing Hormone
  • Thyrotropin-Releasing Hormone
  • Chlordiazepoxide