We used a bioluminescence resonance energy transfer biosensor to screen for functional selective ligands of the human oxytocin (OT) receptor. We demonstrated that OT promoted the direct engagement and activation of G q and all the G i/o subtypes at the OT receptor. Other peptidic analogues, chosen because of specific substitutions in key OT structural/functional residues, all showed biased activation of G protein subtypes. No ligand, except OT, activated G oA or G oB, and, with only one exception, all of the peptides that activated G q also activated G i2 and G i3 but not G i1, G oA, or G oB, indicating a strong bias toward these subunits. Two peptides (DNalOVT and atosiban) activated only G i1 or G i3, failed to recruit β-arrestins, and did not induce receptor internalization, providing the first clear examples of ligands differentiating individual G i/o family members. Both analogs inhibited cell proliferation, showing that a single G i subtype-mediated pathway is sufficient to prompt this physiological response. These analogs represent unique tools for examining the contribution of G i/o members in complex biological responses and open the way to the development of drugs with peculiar selectivity profiles. This is of particular relevance because OT has been shown to improve symptoms in neurodevelopmental and psychiatric disorders characterized by abnormal social behaviors, such as autism. Functional selective ligands, activating a specific G protein signaling pathway, may possess a higher efficacy and specificity on OT-based therapeutics.
It was long believed that G protein-coupled receptors (GPCRs)3 work as bimodal switches between an agonist-promoted “on” state, capable of engaging a specific G protein isoform, and an uncoupled “off” state. However, more recent evidence indicates that GPCR signaling is much more complex than originally thought; a single receptor subtype can activate multiple G protein-dependent and/or G protein-independent effectors, and different agonists can activate the different effectors with different intrinsic efficacies. The most widely accepted theory explaining the signaling complexity of GPCRs is that they adopt a range of distinct conformations that are differentially stabilized or induced by various endogenous or synthetic agonists; this ligand-induced activation of independent signaling conformations has been called “functional selectivity” (1). It has been shown that many ligands acting at GPCRs are characterized by functional selectivity, and the number of “functional selective ligands” has rapidly increased over recent years; however, the structural characteristics underlying functional selectivity are still little understood. In particular, it will be important to determine, at individual GPCRs, the molecular basis of the coupling efficiency of individual ligands to different G protein subtypes and effectors in order to define their potential use in specific cell contexts.
The oxytocin receptor (OTR) is a GPCR whose promiscuous coupling to G q and G i heterotrimeric complexes has been described in several cell types (2–5). In different cell systems, the multiple signaling pathways activated by OTRs may act synergistically (as in the case of the contraction induced in myometrial cells by OTR coupling to Gα q-11 and to the small G proteins of the Rho family) (6). However, they may also have opposite effects on the same cell function, as in the case of neuronal cells in which OT can inhibit (via a PTX-resistant G protein pathway) or stimulate (via a PTX-sensitive G protein pathway) K+ conductances belonging to the inward rectifier family of K+ channels (4). Similarly, in human embryonic kidney HEK293 cells stably transfected with human OTRs, receptor coupling to G i is responsible for inhibiting cell growth, whereas receptor coupling to a pertussis toxin (PTX)-insensitive complex (possibly G q) stimulates cell growth (5, 7).
Because of this heterogeneity in the final outcome of receptor activation, functional selective ligands will be of great help in identifying the roles of the different OTR-elicited pathways in physiological functions; moreover, as they may have distinct therapeutic actions, they may lead to new therapeutic approaches. In the case of OTR-expressing tumors, the activation of specific OTR-G i signaling inhibits cell growth (8) and stops cell migration (9), and in line with these findings, it has been shown that atosiban, identified in our laboratory as a biased agonist that favors G i/o over G q coupling (8), inhibits the growth of human prostate adenocarcinoma cells in vitro (8) and rat and mouse mammary carcinomas in vitro and in vivo (10). The use of OTR-G i functional selective ligands therefore seems to be a promising means of inducing cancer regression and preventing breast and prostate cancer invasion and metastases. Furthermore, it has recently been suggested that the intranasal administration of OT can be used to promote prosocial behavior and decrease anxiety in patients with neurodevelopmental and psychiatric disorders, such as autism and schizophrenia (11, 12). However, the signaling pathways underlying the physiological effects induced by OT in neuronal cells are still largely unknown. The possibility of pharmacologically manipulating OT-induced neurophysiological functions by activating defined signaling cascades should help in the development of innovative OT-based therapeutic protocols.
To develop functional selective OTR ligands and fully exploit their potential, a number of questions concerning the functional coupling of OTRs need to be answered. Which G protein complexes can OTRs couple to? What is the efficiency of OTR coupling to the different G protein complexes? Which pathways can functional selective ligands be effective on? What are the structural features characterizing these analogues? To start answering these questions, we used a bioluminescent resonance energy transfer (BRET)-based biosensor 4 to screen a number of OT/AVP-derived peptides for their ability to activate G q-, G i1-, G i2-, G i3-, G oA-, G oB-, and G s-transducing complexes.
We found that all of the tested AVP and OT analogues harboring substitution in functionally important domains of the peptides activate G q, G i2, and G i3 with comparable relative efficacy, but none of them (not even those that activate the other G i members as effectively as OT) can reliably activate G i1, G oA, and G oB, thus indicating a bias toward these subunits; furthermore, two compounds (DNalOVT and atosiban) were entirely biased toward G i1 or G i3 activation, representing the first examples of biased ligands differentiating G i/o subtypes. We also found that the G i functional selective ligands generated G protein activation without β-arrestin recruitment or OTR internalization, thus indicating that they also have a bias toward β-arrestin activity.
The coelenterazine h came from Molecular Probes, Invitrogen (Milan, Italy), coelenterazine 400a (CLz400) from Biotium (Hayward, CA), and PTX from Sigma-Aldrich. The expression vector for G proteins fused to Renilla luciferase Gα q-97-Rluc, Gα i1-91-Rluc, Gα i2-91-Rluc, Gα i3-91-Rluc, Gα oA-91-Rluc8, Gα oB-91-Rluc8, and Gα s-113-Rluc8 cDNAs are described elsewhere.4 The Gα q, Gα i1, Gα i2, Gα i3, Gα s, Gα oA, Gα oB, and Gγ 2 cDNAs came from the Missouri S&T cDNA Research Center (Rolla, MO). The plasmids encoding GFP 10-Gγ 2 and Gβ 1 have been described previously (13); the plasmids encoding for the human OTR and V 2 R are described in Refs. 14 and 15; and OTR-Rluc, OTR-YFP, and OTR-EGFP are as in Refs. 7 and 16. OTR-GFP 2 was obtained by subcloning the cDNA sequence of the OTR into the pGFP 2-N2 vector (PerkinElmer Life Sciences). Briefly, the OTR in pEGFP-N3 was amplified by PCR using forward (5′-CAAAAAGCTTATGGAGGGCGCGCTCGCAG-3′) and reverse (5′-GTTTGGATCCCGTGGATGGCTGGGAGCAG-3′) primers, and the resulting PCR product was subcloned into the pGFP 2-N2 vector; the construct was confirmed by bidirectional sequencing. The CD4-GFP 10 vector is described elsewhere (17). The expression vector for β-arrestin2-YFP (originally developed in the laboratory of M. Bouvier) came from Dr. J. Perroy (Instítut de Génomique Fonctionelle, Montpellier, France), and the expression vector for β-arrestin1-YFP was from Dr. C. Hoffmann (University of Wuerzburg). β-Arrestin2-Rluc and Rluc-β-arrestin2 have been described previously (18, 19). OT, AVP, AVT, and atosiban came from Bachem (Weil am Rhein, Germany). All of the other peptides used in this study were synthesized as in Refs. 20 and 21.
The DU145 human prostate carcinoma, HEK293, and COS7 cell lines were purchased from the American Type Culture Collection (Manassas, VA). HEK293 cells stably expressing the human OTR cDNA C-terminally fused to EGFP or N-terminally tagged to c-myc have been described elsewhere (5, 7, 22). For transfection, cells were seeded at a density of 3,100,000 cells/well in 100-mm plates on the day before transfection. A mix containing 20 μg of DNA and 60 μg of polyethyleneimine (PEI linear, M r 25,000, Polysciences Europe GmbH, Eppelheim, Germany) was prepared with 1 ml of basic medium (without additives such as serum or antibiotics) and, after 15 min of incubation at room temperature, added directly to cells maintained in 10 ml of complete medium containing 10% FBS. 24 h after transfection, the supplemented DMEM was renewed, and the cells were cultured for a further 24 h before the experiments. 48 h after transfection, the cells were washed twice, detached, and resuspended with PBS, 0.5 m m MgCl 2 at room temperature.
The binding assays were performed at 30 °C on membranes prepared from COS7 cells transiently transfected by means of electroporation with the wild-type human OTR (23, 24), using the radiolabeled OTR receptor agonist [3 H]OT (PerkinElmer Life Sciences); peptide affinities (K i) were determined by means of competition experiments in which the peptide concentrations varied from 10−11 to 10−6 m, and the concentration of the radioligand was 4 × 10−9 m. Nonspecific binding was determined in the presence of unlabeled OT (10−3 m). The ligand binding data (K i) were analyzed by means of non-linear regression, one-site binding competition fit using GraphPad Prism software, version 5 (GraphPad, Inc., San Diego, CA).
To detect and analyze the interactions between OTR and the different Gα subunits by means of BRET 2 experiments, HEK293 cells were co-transfected with OTR-Rluc, GFP 10-Gγ 2, Gβ 1, and one of Gα q, Gα i1, Gα i2, Gα i3, Gα s, Gα oA, or Gα oB. To screen for the effects of the different ligands on G protein activation, HEK293 cells were co-transfected with Gα q-97-Rluc, Gα i1-91-Rluc, Gα i2-91-Rluc, Gα i3-91-Rluc, Gα oA-91-Rluc8, Gα oB-91-Rluc8, and Gα s-113-Rluc8 constructs in the presence of plasmids encoding for GFP 10-Gγ 2, Gβ 1, and the OTR or V 2 R. Finally, to study OTR-mediated β-arrestin recruitment by means of BRET 1 experiments, the cells were co-transfected with OTR-Rluc and β-arrestin1-YFP or β-arrestin2-YFP or with OTR-YFP and β-arrestin2-Rluc or Rluc-β-arrestin2. 48 h after transfection, the cells were washed twice, detached, and resuspended with PBS, 0.5 m m MgCl 2 at room temperature. They were then distributed in a white 96-well microplate (100 μg of proteins/well) (Optiplate, PerkinElmer Life Sciences) and incubated in the presence or absence of different concentrations of OT or different ligands for 2 min before substrate addition. The BRET between Rluc/Rluc8 and GFP 10 was measured immediately after the addition of the Rluc substrate coelenterazine 400a (5 μ m), using an Infinite F500 reader plate (Tecan, Milan, Italy) that allows the sequential integration of light signals detected with
