Decreased levels of internalized thyrotropin-releasing hormone receptors after uncoupling from guanine nucleotide-binding protein and phospholipase-C

Daniel R. Nussenzveig, Marcos Heinflink, Marvin C. Gershengorn

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30 Scopus citations


Internalization of TRH receptor (TRH-R) is dependent on sequences/structures in the receptor carboxyl-terminal tail. Here, we studied whether coupling to guanine nucleotide-binding protein (G-protein) and phospholipase-C (PLC) is involved in internalization. We constructed two mutant TRH-Rs: ∆ 218-263 TRH-R, in which most of the residues that form the putative third intracellular loop were deleted, and D71A TRH-R, in which an Asp in the putative second transmembrane helix was mutated to Ala; these TRH-Rs did not activate PLC when expressed transiently in COS-1 cells. In contrast to wild-type (WT) TRH-Rs, approximately 60% of which were internalized at steady state after binding methyl-HisTRH, only approximately 15% of ∆ 218-263 and D71A TRH-Rs were internalized. Thus, mutant TRH-Rs that do not activate PLC, most likely because they are uncoupled from G-proteins, are internalized to lesser extents than WT TRH-Rs. We also studied the effects of U73122 (1-[6-[[17 β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione), an amino steroid that inhibits receptor-mediated activation of PLC. In COS-1 and AtT-20 cells transfected with WT TRH-Rs and in GH3 cells, U73122 virtually abolished TRH activation of PLC and partially reduced the fraction of WT TRH-Rs internalized. Thus, uncoupling WT TRH-Rs from PLC decreases internalization. We conclude that TRH-R coupling to G-protein and PLC increases the number of TRH-Rs internalized at steady state even though the primary signals for agonist-induced internalization are present in the receptor. These data support the idea that a quaternary complex of TRH/TRH-R/G protein/PLC is normally internalized.

Original languageEnglish (US)
Pages (from-to)1105-1111
Number of pages7
JournalMolecular Endocrinology
Issue number9
StatePublished - Sep 1993


ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology

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