However, we should mention here that a significant difference in = 5, for OTR and = 4 for atosiban; = 4 for OTA2; and = 4 for OTA3), and showing < 0.001). vasopressin V1a (mV1aR) and V1b (mV1bR) subtypes. These three receptors were transiently expressed in vitro for binding and intracellular signaling assays, and then a homology model of the mOTR structure was constructed to investigate how its molecular features compare with human and rat OTR orthologs. Our data show that this selectivity profile of Il6 the natural ligands, OT and AVP, is usually conserved in humans, rats, and mice. Furthermore, we found that the synthetic peptide [Thr4Gly7]OT (TGOT) is usually amazingly selective for the ORY-1001 (RG-6016) mOTR and, like the endogenous OT ligand, activates Gq and Gi and recruits gene expression can be rescued by the activation of cognate vasopressin receptors, thus suggesting that this OT/AVP brain systems have overlapping and/or compensatory functions (Sala et al., 2011). Another level of complexity in developing selective analogs derives from your finding that a single GPCR may couple to more than one G-protein, potentially activating multiple responses. Interestingly, different ligands show different degrees of intrinsic efficacy to different signaling pathways activated by the same receptor, a phenomenon referred to as functional selectivity (Urban et al., 2007; Kenakin, 2011). Because functional selective ligands have been recently explained in the OT/AVP receptor family (in particular for the vasopressin 2 receptor (Jean-Alphonse et al., 2009), OTR (Reversi et al., 2005; Gravati et al., 2010; Busnelli et al., 2012), and V1aR (MacKinnon et al., ORY-1001 (RG-6016) 2009), the screening of the functional selective properties of ligands is becoming a crucial issue for the pharmacological characterization of selective ligands. The aim of this study was to pharmacologically characterize a number of OT/AVP analogs at the OT/AVP receptor subtypes expressed in mouse brain: mOTR, mV1aR, and mV1bR. We found that [Thr4Gly7]OT (TGOT) (Lowbridge et al., 1977) has a amazing selectivity for the mouse OTR through which, like the endogenous OT ligand, it activates Gq and Gi and recruits (GFP10) was fused to Gsubunit expression vector cDNAs came from Missouri S&T cDNA Resource Center (Rolla, MO). The expression vector of luciferase (mOTR-Rluc) was generated by subcloning the entire coding region of mOTR into an Rluc vector (PerkinElmer BioSignal, Inc., Monza, Italy). Cell Cultures. HEK293 and COS7 cells purchased from your American Type Culture Collection (Manassas, VA) were produced in Dulbeccos altered Eagles medium (Sigma-Aldrich, Milan, Italy), supplemented with 10% fetal calf serum and 1% penicillin-streptomycin (Sigma-Aldrich) in a 10% CO2 humidified atmosphere at 37C. Transfection. For the ligand binding assays, the COS7 cells were transfected by means of electroporation as previously explained (Chini et al., 1995). For the homogeneous time-resolved fluorescence (HTRF) and bioluminescence resonance energy transfer (BRET) assays, HEK293 cells were seeded at a density of 3,100,000 cells/well in 100-mm plates on the day before transfection. A mix made up of 20 is the concentration of radioligand used in each experiment and the subunits were analyzed by means of BRET2 experiments that use RLuc as the donor, the DeepBlueC coelenterazine derivative as its substrate, and GFP10 as the acceptor. HEK293 cells were cotransfected with mOTR-Rluc, GFP10-Gtest for the extra sum of squares theory (*< 0.05; **< 0.01; ***< 0.001). Ligand-induced BRET ratios are expressed as mean S.E.M and were analyzed with one-way analysis of variance followed by Tukeys post hoc test to determine statistically significant differences in treatments (***< 0.001). The BRET1 kinetics data were normalized by setting the zero time point immediately after the addition of the ligand, and the data were analyzed by means of nonlinear least-squares fitted to the one-phase exponential association equation. Homology Modeling of the mOTR Structure. A large number of GPCR crystal structures in different activity-state-related conformations have been published in recent years (Zhao and Wu, 2012), most of them cocrystallized with specific ligands (agonists ORY-1001 (RG-6016) or antagonists) (Kobilka and Schertler, 2008; Hanson and Stevens, 2009). Therefore, they serve as optimal templates for family A GPCR homology modeling (OTRs are users of family A GPCRs) with the purpose to study potential details of ligand binding or transmission transduction. Based on high sequence similarity and overlapping structural features in the transmembrane helices (TMHs), the = 3; 1.11 nM 27% CV, = 4; and 0.43 nM 12% CV, = 4), whereas OT experienced a receptor-specific affinity range that ORY-1001 (RG-6016) was highest for OTR (= 4) and lower for V1aR (= 5) (< 0.001 versus mOTR) and V1bR (= 4) (< 0.001 versus mOTR). The dLVT peptide agonist binds with significantly different = 5; 3.39 nM 28% CV, = 5 (< 0.001 versus mOTR); and 0.82 nM 7% CV, = 3 (< 0.01 versus mOTR) (Fig..