title: A constitutively active mutant thyrotropin-releasing hormone receptor is chronically down-regulated in pituitary cells: evidence using chlordiazepoxide as a negative antagonist.
abstract: 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.
authors.affiliation: Department of Medicine, Cornell University Medical College, New York, New York, USA.
authors.forename: M
authors.initials: M
authors.lastname: Heinflink
authors.forename: D R
authors.initials: DR
authors.lastname: Nussenzveig
authors.forename: H
authors.initials: H
authors.lastname: Grimberg
authors.forename: M
authors.initials: M
authors.lastname: Lupu-Meiri
authors.forename: Y
authors.initials: Y
authors.lastname: Oron
authors.forename: M C
authors.initials: MC
authors.lastname: Gershengorn
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002707:Chlordiazepoxide / Q000378:metabolism / Q000494:pharmacology*; D015536:Down-Regulation; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D051379:Mice; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D010911:Pituitary Neoplasms / Q000473:pathology; D011487:Protein Conformation; D018004:Receptors, Bombesin / Q000378:metabolism; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000096:biosynthesis / Q000737:chemistry / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D015290:Second Messenger Systems / Q000187:drug effects; D013973:Thyrotropin-Releasing Hormone / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D007295:Inositol Phosphates; D018004:Receptors, Bombesin; D018025:Receptors, Thyrotropin-Releasing Hormone; D013973:Thyrotropin-Releasing Hormone; D002707:Chlordiazepoxide