To study the effect of agonist on the TRH (thyrotrophin-releasing hormone) receptor protein, an epitope-tagged receptor was stably expressed in HEK-293 cells (human embryonic kidney 293 cells) and receptor levels were measured by immunoblotting. TRH caused a 5–25-fold increase in receptor protein during 48 h, which was half-maximal at 1 nM and was slowly reversible after hormone withdrawal. Chlordiazepoxide, an inverse agonist, had no effect. TRH up-regulation was mimicked by phorbol ester and blocked by the protein kinase C inhibitor GF109203X in combination with thapsigargin, which prevents a calcium response. TRH and phorbol ester increased the density of immunoreactive receptors localized at the cell surface and [3H]MeTRH (where MeTRH stands for [N3-methyl-His]TRH) binding. TRH also increased the concentration of a truncated, internalization-defective receptor. Analysis of cell lines stably expressing TRH receptors fused to the green fluorescent protein on a fluorescence-activated cell sorter showed that TRH and phorbol ester caused 2.7- and 6.8-fold increases in fusion protein expression respectively. TRH receptor up-regulation was only partially accounted for by changes in receptor mRNA, which increased 1.7-fold. TRH caused a small increase in receptor concentration in the presence of cycloheximide, actinomycin D or MG132. In contrast with the results obtained with the TRH receptor, agonist decreased the concentration of stably expressed β2-adrenergic receptors. These results show that TRH increases receptor concentration by a complex mechanism that requires signal transduction but not receptor endocytosis.
Abbreviations used: GFP, green fluorescent protein; HA, haemagglutinin; HEK-293 cells, human embryonic kidney 293 cells; TRH, thyrotrophin-releasing hormone; MeTRH, [N3-methyl-His]TRH; TRHR, TRH receptor.