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/stru ctures in the receptor carboxyl-terminal tail. Here, we studied whethe r coupling to guanine nucleotide-binding protein (G-protein) and phosp holipase-C (PLC) is involved in internalization. We constructed two mu tant TRH-Rs: DELTA218-263 TRH-R, in which most of the residues that fo rm the putative third intracellular loop were deleted, and D71A TRH-R, in which an Asp in the putative second transmembrane helix was mutate d 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 6 0% of which were internalized at steady state after binding methyl-His TRH, only approximately 15% of DELTA218-263 and D71A TRH-Rs were inter nalized. Thus, mutant TRH-Rs that do not activate PLC, most likely bec ause they are uncoupled from G-proteins, are internalized to lesser ex tents than WT TRH-Rs. We also studied the effects of U73122 0)-trien-1 7-yl]amino]hexyl]-1H-pyrrole-2,5-dione), an amino steroid that inhibit s receptor-mediated activation of PLC. In COS-1 and AtT-20 cells trans fected with WT TRH-Rs and in GH3 cells, U73122 virtually abolished TRH activation of PLC and partially reduced the fraction of WT TRH-Rs int ernalized. Thus, uncoupling WT TRH-Rs from PLC decreases internalizati on. 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 recept or. These data support the idea that a quaternary complex of TRH/TRH-R /G protein/PLC is normally internalized.