Journal of Biological Chemistry
Volume 277, Issue 52, 27 December 2002, Pages 50422-50430
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MECHANISMS OF SIGNAL TRANSDUCTION
Homo- and Hetero-oligomerization of Thyrotropin-releasing Hormone (TRH) Receptor Subtypes: DIFFERENTIAL REGULATION OF β-ARRESTINS 1 AND 2*

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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 β-arrestin 1 or 2 promoted TRHR1 internalization. In contrast, only β-arrestin 2 could enhance TRHR2 internalization. The preference for β-arrestin 2 by TRHR2 was supported by the impairment of TRHR2 trafficking in mouse embryonic fibroblasts (MEFs) from either a β-arrestin 2 knockout or a β-arrestin 1/2 knockout, while TRHR1 trafficking was only abolished in MEFs lacking both β-arrestins. The differential β-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 β-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 β-arrestins, could also enhance TRHR2/β-arrestin 1 interaction. This study demonstrates that TRHR subtypes are differentially regulated by the β-arrestins and also provides the first evidence that the interactions of TRHRs with β-arrestin may be altered by hetero-oligomer formation.

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Published, JBC Papers in Press, October 21, 2002, DOI 10.1074/jbc.M209340200

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This work was supported by grants (to K. A. E.) from the National Health & Medical Research Council of Australia (Project Grant ID: 212065), the Raine Foundation, and the Keogh Institute for Medical Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.