Dominic Devost

A Novel Biased Allosteric Compound Inhibitor of Parturition Selectively Impedes the Prostaglandin F2α-mediated Rho/ROCK Signaling Pathway

The prostaglandin F2α (PGF2α) receptor (FP) is a key regulator of parturition and a target for pharmacological management of preterm labor. However, an incomplete understanding of signaling pathways regulating myometrial contraction hinders the development of improved therapeutics. Here we used a peptidomimetic inhibitor of parturition in mice, PDC113.824, whose structure was based on the NH2-terminal region of the second extracellular loop of FP receptor, to gain mechanistic insight underlying FP receptor-mediated cell responses in the context of parturition. We show that PDC113.824 not only delayed normal parturition in mice but also that it inhibited both PGF2α- and lipopolysaccharide-induced preterm labor. PDC113.824 inhibited PGF2α-mediated, Gα12-dependent activation of the Rho/ROCK signaling pathways, actin remodeling, and contraction of human myometrial cells likely by acting as a non-competitive, allosteric modulator of PGF2α binding. In contrast to its negative allosteric modulating effects on Rho/ROCK signaling, PDC113.824 acted as a positive allosteric modulator on PGF2α-mediated protein kinase C and ERK1/2 signaling. This bias in receptor-dependent signaling was explained by an increase in FP receptor coupling to Gαq, at the expense of coupling to Gα12. Our findings regarding the allosteric and biased nature of PDC113.824 offer new mechanistic insights into FP receptor signaling relevant to parturition and suggest novel therapeutic opportunities for the development of new tocolytic drugs.

V1b and CRHR1 Receptor Heterodimerization Mediates Synergistic Biological Actions of Vasopressin and CRH

Vasopressin (AVP) and CRH synergistically regulate adrenocorticotropin and insulin release at the level of the pituitary and pancreas, respectively. Here, we first extended these AVP and CRH coregulation processes to the adrenal medulla. We demonstrate that costimulation of chromaffin cells by AVP and CRH simultaneously induces a catecholamine secretion exceeding the one induced by each hormone alone, thus demonstrating a net potentiation. To further elucidate the molecular mechanisms underlying this synergism, we coexpressed human V1b and CRH receptor (CRHR)1 receptor in HEK293 cells. In this heterologous system, AVP also potentiated CRH-stimulated cAMP accumulation in a dose-dependent and saturable manner. This effect was only partially mimicked by phorbol ester or inhibited by a phospholipase C inhibitor respectively. This finding suggests the existence of an new molecular mechanism, independent from second messenger cross talk. Similarly, CRH potentiated the AVP-induced inositol phosphates production. Using bioluminescence resonance energy transfer, coimmunoprecipitation, and receptor rescue experiments, we demonstrate that V1b and CRHR1 receptors assemble as heterodimers. Moreover, new pharmacological properties emerged upon receptors cotransfection. Taken together, these data strongly suggest that direct molecular interactions between V1b and CRHR1 receptors play an important role in mediating the synergistic interactions between these two receptors.

C5L2 and C5aR interaction in adipocytes and macrophages: Insights into adipoimmunology

Obesity is associated with inflammation characterized by increased infiltration of macrophages into adipose tissue. C5aR-like receptor 2 (C5L2) has been identified as a receptor for acylation-stimulating protein (ASP) and the inflammatory factor C5a, which also binds C5aR. The present study examines the effects of ligands ASP and C5a on interactions between the receptors C5L2 and C5aR in 3T3-L1 adipocytes and J774 macrophages. BRET experiments indicate that C5L2 and C5aR form homo- and heterodimers in transfected HEK 293 cells, which were stable in the presence of ligand. Cell surface receptor levels of C5L2 and C5aR increased during 3T3-L1 adipocyte differentiation; both receptors are also highly expressed in J774 macrophages. Using confocal microscopy to evaluate endogenous receptors in adipocytes following stimulation with ASP or C5a, C5L2 is internalized with increasing perinuclear colocalization with C5aR. There is little C5a-dependent colocalization in macrophages. While adipocyte-conditioned medium (ACM) increased C5L2-C5aR colocalization in macrophages, this was blocked by C5a. ASP stimulation increased Akt (Ser(473)) phosphorylation in both cell types; C5a induced slight Akt phosphorylation in adipocytes with less effect in macrophages. ASP, but not C5a, increased fatty acid uptake/esterification in adipocytes. C5L2-C5aR homodimerization versus heterodimerization may thus contribute to differential responses obtained following ASP vs C5a stimulation of adipocytes and macrophages, providing new insights into the complex interaction between these two cell types within adipose tissue. Studying the mechanisms involved in the differential responses of C5L2-C5aR activation based on cell type will further our understanding of inflammatory processes in obesity.

Identification of interleukin-1β regulated genes in uterine smooth muscle cells

We analyzed the response of uterine smooth muscle cells to interleukin-1beta (IL-1beta). We first showed that PHM1-31 myometrial cells, our cellular model, are contractile. To determine the molecular mechanisms of uterine smooth muscle cell activation by proinflammatory cytokines, we performed genechip expression array profiling studies of PHM1-31 cells in the absence and the presence of IL-1beta. In total, we identified 198 known genes whose mRNA levels are significantly modulated (> 2.0-fold change) following IL-1beta exposure. We confirmed the expression changes for selected genes by independent mRNA and protein analysis. The group of genes induced by IL-1beta includes transcription factors and inflammatory response genes such as nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFkappaB), pentraxin-related gene (PTX3), and tumor necrosis factor alpha-induced protein 3/A20 (TNFAIP3/A20). We also found up-regulation of chemokines like C-X-C motif ligand 3 (CXCL3) and extracellular matrix remodeling signaling molecules like tenascin C (TNC). Our data suggest that IL-1beta elicits the rapid activation of a cellular network of genes particularly implicated in inflammatory response that may create a cellular environment favorable for myometrial cell contraction. Our results provide novel insights into the mechanisms of uterine smooth muscle cell regulation and possibly infection-induced preterm labor.

Functional Activity of the Carboxyl‐Terminally Extended Oxytocin Precursor Peptide During Cardiac Differentiation of Embryonic Stem Cells

The hypothalamic post‐translational processing of oxytocin (OT)‐neurophysin precursor involves the formation of C‐terminally extended OT forms (OT‐X) that serve as intermediate prohormones. Despite abundant expression of the entire functional OT system in the developing heart, the biosynthesis and implication of OT prohormones in cardiomyogenesis remain unknown. In the present work, we investigated the involvement of OT‐X in cardiac differentiation of embryonic stem (ES) cells. Functional studies revealed the OT receptor‐mediated cardiomyogenic action of OT‐Gly‐Lys‐Arg (OT‐GKR). To obtain further insight into the mechanisms of OT‐GKR‐induced cardiac effects, we generated ES cell lines overexpressing the OT‐GKR gene and enhanced green fluorescent protein (EGFP). The functionality of the OT‐GKR/EGFP construct was assessed by fluorescence microscopy and flow cytometry, with further confirmation by radioimmunoassay and immunostaining. Increased spontaneously beating activity of OT‐GKR/EGFP‐expressing embryoid bodies and elevated expression of GATA‐4 and myosin light chain 2v cardiac genes indicated an inductive effect of endogenous OT‐GKR on ES cell‐derived cardiomyogenesis. Furthermore, patch‐clamp experiments demonstrated induction of ventricular phenotypes in OT‐GKR/EGFP‐transfected and in OT‐GKR‐treated cardiomyocytes. Increased connexin 43 protein in OT‐GKR/EGFP‐expressing cells further substantiated the evidence that OT‐GKR modifies cardiac differentiation toward the ventricular sublineage. In conclusion, this report provides new evidence of the biological activity of OT‐X, notably OT‐GKR, during cardiomyogenic differentiation.

Angiotensin II type I and prostaglandin F2α receptors cooperatively modulate signaling in vascular smooth muscle cells

Background: Evidence suggests that FP modulates AT1R physiological responses. Results: In heterologous and native systems, both receptors allosterically modulated each others function. Conclusion: This is likely via the agency of an AT1R/FP heterodimer. Significance: This may have implications in hypertension management. The angiotensin II type I (AT1R) and the prostaglandin F2α (PGF2α) F prostanoid (FP) receptors are both potent regulators of blood pressure. Physiological interplay between AT1R and FP has been described. Abdominal aortic ring contraction experiments revealed that PGF2α-dependent activation of FP potentiated angiotensin II-induced contraction, whereas FP antagonists had the opposite effect. Similarly, PGF2α-mediated vasoconstriction was symmetrically regulated by co-treatment with AT1R agonist and antagonist. The underlying canonical Gαq signaling via production of inositol phosphates mediated by each receptor was also regulated by antagonists for the other receptor. However, binding to their respective agonists, regulation of receptor-mediated MAPK activation and vascular smooth muscle cell growth were differentially or asymmetrically regulated depending on how each of the two receptors were occupied by either agonist or antagonist. Physical interactions between these receptors have never been reported, and here we show that AT1R and FP form heterodimeric complexes in both HEK 293 and vascular smooth muscle cells. These findings imply that formation of the AT1R/FP dimer creates a novel allosteric signaling unit that shows symmetrical and asymmetrical signaling behavior, depending on the outcome measured. AT1R/FP dimers may thus be important in the regulation of blood pressure.

Conformational profiling of the AT1 angiotensin II receptor reflects biased agonism, G protein coupling and cellular context

Here, we report the design and use of G protein-coupled receptor-based biosensors to monitor ligand-mediated conformational changes in receptors in intact cells. These biosensors use bioluminescence resonance energy transfer with Renilla luciferase (RlucII) as an energy donor, placed at the distal end of the receptor C-tail, and the small fluorescent molecule FlAsH as an energy acceptor, its binding site inserted at different positions throughout the intracellular loops and C-terminal tail of the angiotensin II type I receptor. We verified that the modifications did not compromise receptor localization or function before proceeding further. Our biosensors were able to capture effects of both canonical and biased ligands, even to the extent of discriminating between different biased ligands. Using a combination of G protein inhibitors and HEK 293 cell lines that were CRISPR/Cas9-engineered to delete Gαq, Gα11, Gα12, and Gα13 or β-arrestins, we showed that Gαq and Gα11 are required for functional responses in conformational sensors in ICL3 but not ICL2. Loss of β-arrestin did not alter biased ligand effects on ICL2P2. We also demonstrate that such biosensors are portable between different cell types and yield context-dependent readouts of G protein-coupled receptor conformation. Our study provides mechanistic insights into signaling events that depend on either G proteins or β-arrestin.

Allosteric interactions between the oxytocin receptor and the β2-adrenergic receptor in the modulation of ERK1/2 activation are mediated by heterodimerization.

The oxytocin receptor (OTR) and the β(2)-adrenergic receptor (β(2)AR) are key regulators of uterine contraction. These two receptors are targets of tocolytic agents used to inhibit pre-term labor. Our recent study on the nature of OTR- and β(2)AR-mediated ERK1/2 activation in human hTERT-C3 myometrial cells suggested the presence of an OTR/β(2)AR hetero-oligomeric complex (see companion article). The goal of this study was to investigate potential allosteric interactions between OTR and β(2)AR and establish the nature of the interactions between these receptors in myometrial cells. We found that OTR-mediated ERK1/2 activation was attenuated significantly when cells were pretreated with the β(2)AR agonist isoproterenol or two antagonists, propranolol or timolol. In contrast, pretreatment of cells with a third β(2)AR antagonist, atenolol resulted in an increase in OTR-mediated ERK1/2 activation. Similarly, β(2)AR-mediated ERK1/2 activation was strongly attenuated by pretreatment with the OTR antagonists, atosiban and OTA. Physical interactions between OTR and β(2)AR were demonstrated using co-immunoprecipitation, bioluminescence resonance energy transfer (BRET) and protein-fragment complementation (PCA) assays in HEK 293 cells, the latter experiments indicating the interactions between the two receptors were direct. Our analyses suggest physical interactions between OTR and β(2)AR in the context of a new heterodimer pair lie at the heart of the allosteric effects.

Quantifying biased signaling in GPCRs using BRET-based biosensors

There has been a growing appreciation that G protein-coupled receptor (GPCR) functional selectivity (viz. biased signaling), in particular between G protein- and β-arrestin-dependent signaling, can be achieved with specific ligands, and that such directed signaling represents a promising avenue for improving drug efficacy and therapy. Thus, for any given GPCRs it is important to define means to pharmacologically characterize and classify drugs for their propensity to bias signaling. Here we describe an experimental protocol and step-by-step approach to assess functional selectivity between Gαq and β-arrestin-dependent responses using the prototypical angiotensin II (AngII) type 1 receptor (AT1R) expressed in HEK 293 cells. The protocol describes the expression of Bioluminescence Resonance Energy Transfer (BRET) sensors for either Gαq or β-arrestin with AT1R, and the use of the operational model of pharmacological agonism to quantify ligand bias. Such methods are equally applicable to other GPCRs and their downstream signaling effectors.

Designing BRET-based conformational biosensors for G protein-coupled receptors

Ligand-biased signaling is starting to have significant impact on drug discovery programs in the pharmaceutical industry and has reinvigorated our understanding of pharmacological efficacy. As such, many investigators and screening campaigns are now being directed at a larger section of the signaling responses downstream of an individual G protein-coupled receptor. Many biosensor-based platforms have been developed to capture signaling signatures. Despite our growing ability to use such signaling signatures, we remain hampered by the fact that signaling signatures may be particular to an individual cell type and thus our platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. Here, we provide a complementary strategy based on capturing receptor-proximal conformational profiles using intra-molecular BRET-based sensors composed of a Renilla luciferase donor engineered into the carboxy-terminus and CCPGCC motifs which bind fluorescent hairpin arsenical dyes engineered into different positions in intracellular loop 3 of FP, the receptor for PGF2α. We discuss the design and optimization of such sensors for orthosteric and allosteric ligands.