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

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Internalization of TRH receptor (TRH-R) is dependent on sequences/structures in the receptor carboxylterminal 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: Δ 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 Δ218-263 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-[[17β-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
Volume7
Issue number9
StatePublished - 1993

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Thyrotropin Releasing Hormone Receptors
Guanine Nucleotides
Type C Phospholipases
Protein C
Carrier Proteins
GTP-Binding Proteins
Steroid Receptors
COS Cells
Viperidae
Tail

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology, Diabetes and Metabolism

Cite this

Decreased levels of internalized thyrotropin-releasing hormone receptors after uncoupling from guanine nucleotide-binding protein and phospholipase-C. / Nussenzveig, Daniel R.; Heinflink, Marcos; Gershengorn, Marvin C.

In: Molecular Endocrinology, Vol. 7, No. 9, 1993, p. 1105-1111.

Research output: Contribution to journalArticle

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abstract = "Internalization of TRH receptor (TRH-R) is dependent on sequences/structures in the receptor carboxylterminal 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: Δ 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 Δ218-263 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-[[17β-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.",
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