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To investigate the function of dimerization of the TRH receptor, a controlled dimerization system was developed. A variant FK506 binding protein (FKBP) domain was fused to the receptor C terminus and dimerization induced by incubating cells with dimeric FKBP ligand, AP20187. The TRH receptor-fusion bound hormone and signaled normally. Addition of dimerizer to cells expressing the receptor-FKBP fusion dramatically increased the fraction of receptor running as dimer on SDS-PAGE. AP20187 caused dimerization in a time- and concentration-dependent manner, acting within 1 min. Dimerizer had no effect on TRH receptors lacking the FKBP domain, and its effects were blocked by excess monomeric FKBP ligand. AP20187-induced dimerization did not cause receptor phosphorylation, inositol phosphate production, or ERK1/2 activation, and dimerizer did not alter signaling by TRH. Induced dimerization did, however, alter TRH receptor trafficking. TRH promoted greater receptor internalization in cells treated with AP20187 but not monomeric ligand, based on loss of surface binding sites and immunostaining. Dimerization increased the rate of internalization of TRH receptors and decreased the apparent rate of receptor recycling. AP20187 enhanced the small amount of TRH-induced receptor internalization when the receptor-FKBP fusion protein was expressed in cells lacking beta-arrestins. The results show that controlled dimerization of the TRH receptor potentiates hormone-induced receptor trafficking.

These studies were designed to characterize ubiquitination of the G protein-coupled TRH receptor (TRHR). TRHRs and ubiquitin coprecipitated with antibodies to either receptor or ubiquitin in Chinese hamster ovary or pituitary GHFT cells. Inhibition of the proteasome with MG-132 resulted in an accumulation of total TRHRs and the appearance of a small amount of cytosolic receptor. MG-132 caused an increase in newly synthesized receptors, detected by microscopy using a TRHR coupled to Timer, a DsRed that undergoes a spontaneous time-dependent color change. Misfolded TRHRs were particularly heavily ubiquitinated. These results show that the proteasome participates in TRHR quality control early after receptor synthesis. Under normal circumstances, most ubiquitinated TRHRs were absorbed to wheat germ agglutinin, indicating that they had undergone complex glycosylation in the Golgi apparatus. When cells were treated with tunicamycin to block glycosylation, a ladder of ubiquitinated species was detectable. Cell surface receptors, which were labeled selectively with either radioligand or antibody, showed no detectable ubiquitin modification. To determine if ubiqutination plays a role in TRH-induced receptor endocytosis, the receptor was expressed in Ts20 cells, which have a temperature-sensitive ubiquitin pathway. TRH induced a significant calcium response and rapid and extensive receptor internalization at both the permissive and nonpermissive temperatures, indicating that ligand-dependent ubiquitination of the receptor, or any other protein, is not necessary for TRHR signaling or internalization. These results show that ubiquitin modification targets misfolded receptors for degradation and suggest a possible role for ubiquitination in receptor trafficking.

PRL synthesis by GH cells in culture has previously been shown to increase when calcium is added to cultures grown in calcium-depleted medium or when cultures are treated for 18 h or longer with the dihydropyridine calcium channel agonist BAY K8644, whereas the antagonist nimodipine inhibits PRL. The experiments described here were designed to test whether differences in PRL synthesis caused by the dihydropyridines are due to changes in PRL mRNA levels, whether structurally different classes of calcium channel blockers alter PRL production, and whether long term treatment with calcium channel agonists and antagonists alters intracellular free calcium, [Ca2+]i. PRL synthesis and PRL mRNA levels were increased similarly by BAY K8644 and decreased in parallel by the dihydropyridine antagonist nimodipine, while overall protein and RNA synthesis were not changed by either the agonist or antagonist. Two calcium channel blockers which act at different sites on L-type channels than the dihydropyridines also inhibited PRL synthesis without affecting GH; 5 microM verapamil reduced PRL by 64% and 15 microM diltiazem by 89%. Partial depolarization with 5-25 mM KCl increased PRL synthesis up to 2-fold. The intracellular free calcium ion concentration was estimated by Quin 2 and averaged 142 nM for control cultures in normal medium, and 128 and 168 nM for cultures treated 72 h with nimodipine or BAY K8644, respectively. Nimodipine totally prevented the calcium rise obtained upon depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)