Homo- and Hetero-oligomerization of Thyrotropin-releasing Hormone (TRH) Receptor Subtypes

Abstract

G-protein-coupled receptors (GPCRs) are regulated by a complex network of mechanisms such as oligomerization and internalization. Using the GPCR subtypes for thyrotropin-releasing hormone (TRHR1 and TRHR2), the aim of this study was to determine if subtype-specific differences exist in the trafficking process. If so, we wished to determine the impact of homo-and hetero-oligomerization on TRHR subtype trafficking as a potential mechanism for the differential cellular responses induced by TRH. Expression of either ␤-arrestin 1 or 2 promoted TRHR1 internalization. In contrast, only ␤-arrestin 2 could enhance TRHR2 internalization. The preference for ␤-arrestin 2 by TRHR2 was supported by the impairment of TRHR2 trafficking in mouse embryonic fibroblasts (MEFs) from either a ␤-arrestin 2 knockout or a ␤-arrestin 1/2 knockout, while TRHR1 trafficking was only abolished in MEFs lacking both ␤-arrestins. The differential ␤-arrestin-dependence of TRHR2 was directly measured in live cells using bioluminescence resonance energy transfer (BRET). Both BRET and confocal microscopy were also used to demonstrate that TRHR subtypes form hetero-oligomers. In addition, these hetero-oligomers have altered internalization kinetics compared with the homo-oligomer. The formation of TRHR1/2 heteromeric complexes increased the interaction between TRHR2 and ␤-arrestin 1. This may be due to conformational differences between TRHR1/2 hetero-oligomers versus TRHR2 homo-oligomers as a mutant TRHR1 (TRHR1 C335Stop) that does not interact with ␤-arrestins, could also enhance TRHR2/␤-arrestin 1 interaction. This study demonstrates that TRHR subtypes are differentially regulated by the ␤-arrestins and also provides the first evidence that the interactions of TRHRs with ␤-arrestin may be altered by hetero-oligomer formation. G-protein-coupled receptor (GPCR)1 function is underpinned by the formation of protein-protein interactions and complexes that are dynamically regulated by a variety of stimuli. These molecular interactions are involved in receptor recognition, activation, and desensitization and are targets for the majority of current therapeutic compounds utilized to treat a variety of disorders. More recently, the escalating body of evidence for GPCR oligomerization also adds another level of complexity in understanding how GPCRs are activated and signal and traffick in the cell. GPCR hetero-oligomerization may explain the diverse physiological responses obtained from a single ligand. Indeed, a number of GPCR subtypes such as the somatostatin, opioid, angiotensin, and ␤-adrenergic receptors have been shown to form hetero-oligomers that result in a receptor unit with either altered ligand specificity, signaling, or internalization rates (1-5). GPCR activation is also regulated by receptorprotein interactions involved in desensitization and internalization. For the majority of GPCRs, the mechanism of internalization is via the ␤-arrestin and dynamin-dependent pathway where agonist-activated phosphorylated receptors interact with the ␤-arres...

The structural basis of arrestin-mediated regulation of G-protein-coupled receptors

“…The formation of TRHR1/2 heterodimers increased the interaction of TRHR2 with arrestin2. Most importantly, even heterodimerization between TRHR2 and truncated TRHR1 (C335Stop), which by itself does not interact with non-visual arrestins, enhanced TRHR2-arrestin2 interaction (Hanyaloglu et al, 2002). The simplest mechanistic interpretation of these findings is that in a heterodimer with TRHR1 (but not in the TRHR2 homo-dimer) the conformation of the TRHR2 cytoplasmic elements changes in such a way as to permit arrestin2 binding tight enough to promote arrestin2-dependent internalization.…”

“…However, a few recent studies suggest that at least non-visual arrestins can bind receptors within a dimer. One study took advantage of the difference between arrestin preference of the 2 types of thyrotropin-releasing hormone receptor (TRHR): TRHR1 interacts with both arrestin2 and arrestin3, whereas TRHR2 interacts only with arrestin3, as evidenced by the enhanced internalization of TRHR1 by overexpression of either non-visual arrestin; TRHR2 endocytosis was enhanced only by arrestin3 (Hanyaloglu et al, 2002). Accordingly, the trafficking of TRHR2 was impaired in mouse embryonic fibroblasts lacking either arrestin3 or both arrestins 2 and 3, whereas the trafficking of TRHR1 was only impaired in fibroblasts lacking both non-visual arrestins.…”

The Natural Inverse Agonist Agouti-related Protein Induces Arrestin-mediated Endocytosis of Melanocortin-3 and -4 Receptors

“…First, using the BRET technique, we provide evidence that binding of ␣-MSH to the MC3R induced the recruitment of ␤-arrestins in living cells. BRET assays have been successfully used to monitor arrestinreceptor interactions of multiple GPCRs (25,26,36,37) and thus have become an established tool to analyze the interaction of a given receptor with arrestins in living cells. Second, cotransfection of siRNAs specifically decreasing expression of ␤-arrestins reduced MC3R endocytosis in HEK-293 cells.…”

“…To address this question, we monitored recruitment of ␤-arrestin-1-YFP to MCR-Rluc fusion proteins in HEK-293 cells using the BRET technique as described in previous reports (25,26,36,37). As shown in Fig.…”

Dimerization of the thyrotropin-releasing hormone receptor potentiates hormone-dependent receptor phosphorylation

“…Inhibitors of receptor homodimerization and dimerization-defective mutants are not available for most GPCRs, including the TRH receptor. Although dimerization of the TRH receptor has been demonstrated by several approaches and TRH has been found to increase the fraction of receptors behaving as dimers (14,15), the physiological ratio of the monomer:dimer:higher oligomer in cells is unknown. Solubilized TRH receptors run at the size of monomers and dimers on SDS/PAGE, and receptors with different epitope tags coimmunoprecipitate when coexpressed (14).…”