Thyrotropin-Releasing Hormone Receptor Processing: Role of Ubiquitination and Proteasomal Degradation

Abstract

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.

The Lysophosphatidic Acid 2 Receptor Mediates Down-regulation of Siva-1 to Promote Cell Survival

“…Thus far, a number of G protein-coupled receptors such as the ␤ 2 -adrenergic receptor, the CXCR4 chemokine receptor, the protease-activated receptor 2 and the V2 vasopressin receptor have been shown to undergo ubiquitination and degradation following prolonged agonist stimulation (39 -42). In contrast, several other receptors such as the plateletactivating factor receptor, the opioid receptor, and the thyrotropin-releasing hormone receptor are ubiquitinated in an agonist-independent manner because of misfolding or incomplete folding of the receptor during synthesis (42)(43)(44). Our results show that the LPA 2 receptor is ubiquitinated in the absence of ligand; however, in the presence of MG-132, LPA stimulation further promotes ubiquitination of the LPA 2 receptor.…”

G Protein-Coupled Receptor Trafficking in Health and Disease: Lessons Learned to Prepare for Therapeutic Mutant Rescue in Vivo

“…In the case of particular proteins (e.g., the GnRHR, the V2R, and rhodopsin), this approach has succeeded with a striking number of different mutants, supporting the view that pharmacoperones will become powerful ammunition in our therapeutic arsenal (Bernier et al, 2004b). On the other hand, it has also become clear that variable amounts of even some WT GPCRs are misrouted, presumably as a result of misfolding , 2001Andersson et al, 2003;Janovick et al, 2003b;Lu et al, 2003Lu et al, , 2004Cook et al, 2003;Pietilä et al, 2005), suggesting that this level of post-translational control may itself be amenable to pharmacological intervention and provide another level of potential therapeutic intervention (UlloaAguirre et al, 2006).…”

“…The further observation that pharmacoperones increase the PME of the WT human GnRHR itself, but not the rat counterpart, means that the human protein was only partially transferred to the PM and the remainder was apparently retained in the ER and eventually degraded, as has been found for other intracellularly retained GPCRs Andersson et al, 2003;Cook et al, 2003;Lu et al, 2003). Moreover, the observation that the human GnRHR is more susceptible to mutations than the rat or mouse ortholog supports the view that the human receptor is very precariously balanced between retention in the ER and routing to the PM and provides an underlying mechanism for a novel level of post-translational regulation of WT proteins (Conn et al, 2006a,b;Janovick et al, 2006).…”

“…Moreover, the observation that the human GnRHR is more susceptible to mutations than the rat or mouse ortholog supports the view that the human receptor is very precariously balanced between retention in the ER and routing to the PM and provides an underlying mechanism for a novel level of post-translational regulation of WT proteins (Conn et al, 2006a,b;Janovick et al, 2006). Retention of WT proteins is sometimes referred to as "inefficient" protein utilization and there is evidence that this mechanism is used by a variety of systems (Petäjá -Repo et al, 2001;Andersson et al, 2003;Cook et al, 2003;Lu et al, 2003). It is not seen in rat or mouse GnRHRs, which route very efficiently to the PM (Janovick et al, 2003b), suggesting that the expression of wild-type receptors that route heavily to the PM and are not retained by the ER QCS normally may be less sensitive to point mutations, as exemplified by the rat and mice GnRHRs.…”

Role of the proteasome in modulating native G-CSFR expression

“…For these experiments, confocal immunofluorescence microscopy was utilized and methods similar to those previously described for the growth hormone receptor (GHR) and the thyrotropin-releasing hormone receptor (TRHR) [17,32]. As shown in Figure 3 (upper panels, A-C), at the permissive temperature of 30°C, the WT G-CSFR was rapidly internalized after ligand-binding as indicated by the vesicular fluorescent intracellular distribution pattern that was apparent within 30 min.…”