title: Homo- and hetero-oligomerization of thyrotropin-releasing hormone (TRH) receptor subtypes. Differential regulation of beta-arrestins 1 and 2.
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.
authors.affiliation: 7TM Receptor Laboratory, Western Australian Institute for Medical Research (WAIMR), University of Western Australia, Centre for Medical Research, Nedlands, Perth, WA 6009, Australia.
authors.forename: Aylin C
authors.initials: AC
authors.lastname: Hanyaloglu
authors.forename: Ruth M
authors.initials: RM
authors.lastname: Seeber
authors.forename: Trudy A
authors.initials: TA
authors.lastname: Kohout
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Lefkowitz
authors.forename: Karin A
authors.initials: KA
authors.lastname: Eidne
mesh_terms: D000818:Animals; D019390:Arrestins / Q000737:chemistry / Q000378:metabolism*; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D005347:Fibroblasts / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007668:Kidney; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D046911:Macromolecular Substances; D051379:Mice; D018345:Mice, Knockout; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605255:Arrb1 protein, mouse; C000605256:Arrb1 protein, rat; C000605258:Arrb2 protein, mouse; C000605257:Arrb2 protein, rat; D019390:Arrestins; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D020033:Protein Isoforms; D018025:Receptors, Thyrotropin-Releasing Hormone; D011993:Recombinant Fusion Proteins; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins