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Transmembrane Domain IV of the Gallus gallus VT2 Vasotocin Receptor is Essential for Forming a Heterodimer with the Corticotrophin Releasing Hormone Receptor
Author(s) -
Mikhailova Marina Victorovna,
Blansett Jonathan,
Jacobi Sandie,
Mayeux Philip R.,
Cornett Lawrence E.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.723.5
Subject(s) - förster resonance energy transfer , vasotocin , receptor , chemistry , transmembrane domain , biophysics , yellow fluorescent protein , g protein coupled receptor , microbiology and biotechnology , biology , biochemistry , fluorescence , gene , neuropeptide , physics , quantum mechanics
Corticotropin releasing hormone receptor (CRHR) and the VT 2 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, 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 that transmembrane domains (TMs) as likely sites of the interaction. In this study, we constructed chimerical VT2Rs, tagged with either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), by replacing the 4th transmembrane region (TM4) of VT2R with TM4 of the β 2 ‐adrenergic receptor (β 2 AR). The VT2R/β 2 AR‐chimera was expressed in HeLa cells and membrane trafficking confirmed using confocal microscopy. VT2R/β 2 AR‐chimera functionality was demonstrated by the mobilization of intracellular Ca 2+ with the addition of AVT. FRET analysis was then performed on VT2R/β 2 AR‐chimera/CRHR pairs, and the calculated distance was observed to be >10 nm apart, indicating that heterodimerization was disrupted by mutating TM4. Therefore, TM4 is an essential part of the dimerization interface between the VT2R and CRHR.