Decreased levels of internalized thyrotropin-releasing hormone receptors after uncoupling from guanine nucleotide-binding protein and phospholipase-C.
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: delta 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 delta 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 beta-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.
Nussenzveig
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Department of Medicine, Cornell University Medical College, New York Hospital, New York 10021.
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D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004963:Estrenes / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010902:Pituitary Gland / Q000378:metabolism; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011760:Pyrrolidinones / Q000494:pharmacology; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D017384:Sequence Deletion; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004963:Estrenes; D007295:Inositol Phosphates; D011760:Pyrrolidinones; D018025:Receptors, Thyrotropin-Releasing Hormone; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C074854:U 73343; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate