title: Transmembrane domain IV of the Gallus gallus VT2 vasotocin receptor is essential for forming a heterodimer with the corticotrophin releasing hormone receptor.
abstract: Corticotropin releasing hormone receptor (CRHR) and the VT2 arginine vasotocin receptor (VT2R) are vital links in the hypothalamic-pituitary-adrenal axis that enable a biological response to stressful stimuli in avian species. CRHR and VT2R are both G-protein coupled receptors (GPCRs), and have been shown by us to form a heterodimer via fluorescent resonance energy transfer (FRET) analysis in the presence of their respective ligands, corticotrophin releasing hormone (CRH) and arginine vasotocin (AVT). The dimerization interface of the heterodimer is unknown, but computational analyses predict transmembrane domains (TMs) as likely sites of the interaction. We constructed chimerical VT2Rs, tagged at the C-terminal ends with either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), by replacing the fourth transmembrane region (TM4) of VT2R with TM4 of the beta2-adrenergic receptor (beta2AR). The VT2R/beta2AR chimeras were expressed in HeLa cells and proper trafficking is confirmed by observing cell membrane localization using confocal microscopy. VT2R/beta2AR-YFP chimera functionality was confirmed with a Fura-2 acetoxymethyl ester (Fura-2AM) assay. FRET analysis was then performed on VT2/beta2AR-chimera/CRHR pairs, and the calculated distance was observed to be >10 nm apart, indicating that heterodimerization was partly disrupted by mutating TM4 of the VT2R. Therefore, TM4 may form one region of the possible dimerization interfaces between the VT2R and CRHR.
authors.affiliation: University of Arkansas for Medical Sciences, Department of Physiology and Biophysics, 4301 West Markham Street, Little Rock, Arkansas 72205, USA. mikhailovamarinav@uams.edu
authors.forename: Marina V
authors.initials: MV
authors.lastname: Mikhailova
authors.forename: Jonathan
authors.initials: J
authors.lastname: Blansett
authors.forename: Sandie
authors.initials: S
authors.lastname: Jacobi
authors.forename: Philip R
authors.initials: PR
authors.lastname: Mayeux
authors.forename: Lawrence E
authors.initials: LE
authors.lastname: Cornett
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002645:Chickens / Q000378:metabolism*; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018019:Receptors, Corticotropin-Releasing Hormone / Q000737:chemistry* / Q000378:metabolism*; D017483:Receptors, Vasopressin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D018019:Receptors, Corticotropin-Releasing Hormone; D017483:Receptors, Vasopressin; C034862:vasotocin receptor