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 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 (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 β 2 -adrenergic receptor (β 2 AR). The VT2R/β 2 AR chimeras were expressed in HeLa cells and proper trafficking is confirmed by observing cell membrane localization using confocal microscopy. VT2R/β 2 AR-YFP chimera functionality was confirmed with a Fura-2 acetoxymethyl ester (Fura-2AM) assay. FRET analysis was then performed on VT2 / β 2 AR-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.

Making Structural Sense of Dimerization Interfaces of Delta Opioid Receptor Homodimers

“…These findings were used to guide a total of 3 μs of well-tempered metadynamics CG simulations to characterize at the atomic level the putative interfaces between interacting protomers of the DOR. In accordance with predictions from correlated mutation analysis of ORs , , and with published experimental evidence for several GPCR dimers and/or oligomers − , we first focused on the TM4 and TM5 interfaces. We discovered that endogenous cysteines at positions 4.48 and 5.41 were cross-linked by HgCl 2 but not by CuP, consistent with the ability of HgCl 2 to penetrate deeper in the membrane and also with its ability to bridge more distant cysteines that might not collide directly.…”

“…Here, we have investigated possible interfaces in DOR homodimeric complexes by means of an integrated experimental−computational approach aimed at selecting energetically favorable dimeric models among all different possibilities amenable to TM4 or TM5 helices. Our focus on these helices was based on a large body of literature suggesting a direct primary involvement of lipid-exposed surfaces of TM4 and/or TM5 in the dimerization/oligomerization interfaces of several GPCRs, in particular: dopamine D2 receptor (D2R) homodimer − , serotonin 5-HT4 receptor homodimer , serotonin 5-HT2C homodimer , α1b-adrenoceptor homodimer , , C5a receptor homodimer , chemokine CCR5 homodimer , serotonin 5-HT2A-metabotropic glutamate receptor 2 heterodimer , secretin , and corticotropin releasing hormone-VT2 arginine vasotocin receptor heterodimer . We performed bioluminescence resonance energy transfer (BRET) experiments to confirm DOR interactions in our experimental system, as well as subsequent cross-linking experiments on DOR constructs with substituted cysteines at the extracellular ends of TM4 and TM5.…”

Lessons from Free Energy Simulations of δ-Opioid Receptor Homodimers Involving the Fourth Transmembrane Helix

“…Here, we performed coarse-grained (CG) umbrella sampling molecular dynamics (MD) simulations of mouse δOR in an explicit palmitoyloleoylphosphatidylcholine (POPC) and 10% cholesterol/water environment to obtain estimates of the free energy difference between δOR monomers and homodimers involving the fourth transmembrane (TM) helix, from which to derive lifetime predictions. The focus on TM4 is justified by a number of published experimental studies on several GPCRs − , including our early correlated mutation analyses of opioid receptor sequences , , suggesting a direct primary association of lipid-exposed surfaces of these helices.…”