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 beta-arrestin 1 or 2 promoted TRHR1 internalization. In contrast, only beta-arrestin 2 could enhance TRHR2 internalization. The preference for beta-arrestin 2 by TRHR2 was supported by the impairment of TRHR2 trafficking in mouse embryonic fibroblasts (MEFs) from either a beta-arrestin 2 knockout or a beta-arrestin 1/2 knockout, while TRHR1 trafficking was only abolished in MEFs lacking both beta-arrestins. The differential beta-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 beta-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 beta-arrestins, could also enhance TRHR2/beta-arrestin 1 interaction. This study demonstrates that TRHR subtypes are differentially regulated by the beta-arrestins and also provides the first evidence that the interactions of TRHRs with beta-arrestin may be altered by hetero-oligomer formation.
Highlights
G-protein-coupled receptors (GPCRs) are regulated by a complex network of mechanisms such as oligomerization and internalization
While expression levels for TRH receptor 1 (TRHR1) were ϳ2.3-fold less than that observed for TRHR2 (Bmax values: TRHR1 287,902 receptor sites/cell; TRHR2 668,564 receptor sites/cell), the maximal level of TRHR1 internalization was greater than that measured for TRHR2 (Fig. 1A), and measurements of half-time rates of internalization (t1⁄2) showed a much faster rate for TRHR1 when compared with TRHR2 in both cell lines (Fig. 1B)
The relatively shorter t1⁄2 values of both TRHR subtypes observed in HEK293 cells may be due to the higher endogenous levels of -arrestins in these cells compared with COS cells, known to express only low levels of -arrestins [42]
Summary
G-protein-coupled receptors (GPCRs) are regulated by a complex network of mechanisms such as oligomerization and internalization. The differential -arrestin-dependence of TRHR2 was directly measured in live cells using bioluminescence resonance energy transfer (BRET) Both BRET and confocal microscopy were used to demonstrate that TRHR subtypes form hetero-oligomers. TRHR1 and 2 are expressed in distinct compartments of the brain and spinal cord [12, 14, 17, 18], areas have been defined where both receptor subtypes are found [12, 14] This raises the question regarding the mechanism of selectivity and receptor regulation used when two TRHR, thyrotropin-releasing hormone receptor; BRET, bioluminescence resonance energy transfer, MEF; mouse embryonic fibroblast; -arr-KO, -arrestin knockout; EYFP, enhanced yellow fluorescent protein; WT, wild type; GFP, green fluorescent protein; IP, inositol phosphate; Rluc, Renilla luciferase. Our findings suggest that TRHR oligomerization results in the creation of novel receptor units that can be differentially regulated by -arrestins
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