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The role of different G protein-coupled receptors (GPCRs) in the cardiovascular system is well understood in cardiomyocytes and vascular smooth muscle cells (VSMCs). In the former, stimulation of Gs-coupled receptors leads to increases in contractility, whereas stimulation of Gq-coupled receptors modulates cellular survival and hypertrophic responses. In VSMCs, stimulation of GPCRs also modulates contractile and cell growth phenotypes. Here, we will focus on the relatively less well-studied effects of GPCRs in cardiac fibroblasts, focusing on key signaling events involved in the activation and differentiation of these cells. We also review the hierarchy of signaling events driving the fibrotic response and the communications between fibroblasts and other cells in the heart. We discuss how such events may be distinct depending on where the GPCRs and their associated signaling machinery are localized in these cells with an emphasis on nuclear membrane-localized receptors. Finally, we explore what such connections between the cell surface and nuclear GPCR signaling might mean for cardiac fibrosis. [ABSTRACT FROM AUTHOR]

The urotensinergic system, comprised of a G protein-coupled receptor (UT) and two endogenous ligands named urotensin II (UII) and urotensin II-related peptide (URP), has garnered significant attention due to its involvement in the initiation and/or the evolution of various diseases. Accordingly, multiple studies using animal models have demonstrated that UT antagonists may have utility as potential therapeutic agents for treating atherosclerosis, pulmonary arterial hypertension, heart failure, and cancer. Unfortunately, clinical investigations of UT antagonist candidates showed limited efficacy in humans. This system, which has yet to be effectively targeted, therefore remains to be therapeutically exploited. Here, we discuss various hypotheses that could explain the in vivo failure of UT antagonists. Structural and sequence differences between rat and human urotensinergic system introduce pharmacological diversity through selection/stabilization of particular UT active conformations that may be ligand- and species-specific. Several reports indicated that UII and URP can exert distinct and separate actions, thereby highlighting the possibility that altered UT signaling in response to UII or URP could lead to distinct roles for each peptide in the pathogenesis and progression of UT-associated diseases. Despite the discovery of URP more than 10 years ago, only a small numbers of UT antagonists have been systematically assessed for their ability to block URP-mediated action. Such studies led to identification of several molecules, both peptidic and nonpeptidic, exerting probe-dependent action with a wide range of activity against UII- and URP-mediated functions. Analysis of currently available UT antagonists may reveal their propensity to trigger the activation of certain subsets of UT-associated signaling as well as exert probe-dependent action upon UII or URP binding. A better understanding of the molecular and cellular pharmacology of the urotensinergic system, including the functional selectivity of its endogenous ligands and its interspecies variability, should facilitate the design of innovative (bias agonists and allosteric modulators) and therapeutically relevant compounds for the treatment of UT-associated diseases. [ABSTRACT FROM AUTHOR]

[Display omitted] The urotensinergic system, involved in the development and/or progression of numerous pathological conditions, is composed of one G protein-coupled receptor (UT) and two endogenous ligands known as urotensin II (UII) and urotensin II-related peptide (URP). These two structurally related hormones, which exert common and divergent effects, are thought to play specific biological roles. In recent years, we have characterized an analog termed urocontrin A (UCA), i.e. [Pep4]URP, which is capable of discriminating the effects of UII from URP. Such an action could allow the delineation of the respective functions of these two endogenous ligands. In an effort to define the molecular determinants involved in this behavior and to improve the pharmacological profile of UCA, we introduced modifications from urantide, considered for some time as a lead compound for the development of UT antagonists, into UCA and assessed the binding, contractile activity and G protein signaling of these newly developed compounds. Our results show that UCA and its derivatives exert probe-dependent effects on UT antagonism, and we have further identified [Pen2, Pep4]URP as a G q biased ligand with an insurmountable antagonism in our aortic ring contraction assay. [ABSTRACT FROM AUTHOR]

Abstract: Urotensin II (UII) and its receptor UT, are widely expressed in the cardiovascular and central nervous system, where they exert regulatory actions under both physiological and pathological conditions. Our study, aimed at investigating the presence of functional nuclear UT in various rat and monkey tissues as well as in human cell lines, demonstrated for the first time by Western blot analysis and confocal immunofluorescence a tissue-specific nuclear expression of this receptor (heart and central nervous system). This nuclear UT was further characterized pharmacologically through radioligand binding studies using specific ligands of the urotensinergic system, as well as somatostatin. In 2D-gel experiments, we observed the presence of different post-translational modifications between membrane and nuclear UT receptors in brain extracts. Transcription initiation assays showed de novo RNA synthesis caused by UII and Urotensin-related peptide (URP) which were inhibited by an UT antagonist urantide. In hypoxic/ischemic conditions, UT receptors were differentially modulated in regard to subcellular localization. Thus, the unique regiospecificity of the nuclear UT receptor along with its particular modulation under hypoxic conditions could indicate a specific and complementary physiological role that could be correlated with pro-angiogenic and/or neuromodulatory actions of UII, both in the cardiovascular and central nervous system. [Copyright &y& Elsevier]

Over the last decade, the urotensinergic system, composed of one G protein-coupled receptor and two endogenous ligands, has garnered significant attention as a promising new target for the treatment of various cardiovascular diseases. Indeed, this system is associated with various biomarkers of cardiovascular dysfunctions and is involved in changes in cardiac contractility, fibrosis, and hypertrophy contributing, like the angiotensinergic system, to the pathogenesis and progression of heart failure. Significant investment has been made toward the development of clinically relevant UT ligands for therapeutic intervention, but with little or no success to date. This system therefore remains to be therapeutically exploited. Pepducins and other lipidated peptides have been used as both mechanistic probes and potential therapeutics; therefore, pepducins derived from the human urotensin II receptor might represent unique tools to generate signaling bias and study hUT signaling networks. Two hUT-derived pepducins, derived from the second and the third intracellular loop of the receptor (hUT-Pep2 and [Trp¹, Leu²]hUT-Pep3, respectively), were synthesized and pharmacologically characterized. Our results demonstrated that hUTPep2 and [Trp¹, Leu²]hUT-Pep3 acted as biased ago-allosteric modulators, triggered ERK1/2 phosphorylation and, to a lesser extent, IP1 production, and stimulated cell proliferation yet were devoid of contractile activity. Interestingly, both hUTderived pepducins were able to modulate human urotensin II (hUII)- and urotensin II-related peptide (URP)-mediated contraction albeit to different extents. These new derivatives represent unique tools to reveal the intricacies of hUT signaling and also a novel avenue for the design of allosteric ligands selectively targeting hUT signaling potentially. [ABSTRACT FROM AUTHOR]

The pituitary adenylate cyclase-activating polypeptide (PACAP), which exists in two isoforms of 27 and 38 amino acids, can induce neuronal protection in vitro and in vivo following the activation of PAC1, a class B G protein-coupled receptor (GPCR). With its potent neuroprotective and anti-inflammatory effects, this peptide represents a promising avenue for the development of therapeutic strategies to potentially cure or at least slow the progression of neurodegenerative disorders. Beyond the canonical G protein signal effectors, GPCRs are also coupled to a multitude of intracellular signaling pathways that can be independently activated by biased ligands, thereby expanding vastly the potential for discovering new drugs. Interestingly, some studies have demonstrated distinct signaling features for the PACAP isoforms. With this observation in mind, we assessed the impact of chemical and structural modifications introduced into specific regions of the PACAP isoforms on their neuroprotective effects, and determined the role played by these physico-chemical and structural features on their signaling signatures. Each compound was also evaluated for its ability to bind the PACAP receptors, promote cell survival in a cellular model of Parkinson’s disease and stimulate the signaling partners associated with PAC1 activation, including G s and G q , as well as β-arrestin 1 and 2. Our results demonstrate that PACAP38 and its related analogs exert a more potent neuroprotective action than their 27-amino acid counterparts and that this neuroprotective effect is dependent on both G q and G s -dependent signaling. This study will definitely improve our understanding of the molecular and cellular mechanisms associated with PACAP neuroprotection. [ABSTRACT FROM AUTHOR]

While sharing common biological activity, the two endogenous ligands of the G protein-coupled receptor UT, e.g. urotensin II (UII) and urotensin II-related peptide (URP), also exhibit distinct effects that could be explained by distinct interactions with their cognate receptor (UT). Accordingly, introduction of a similar substitution at the intracyclic Tyr residue in UII and URP led to compounds with divergent pharmacologic profiles. Hypothesizing that the Tyr 6 residue of URP is a key-element to understand the specific activation of UT by URP, we undertook a study of the structure-activity relationship in which this particular residue was replaced by non-natural and constrained amino acids. Each compound was evaluated for its ability to bind UT, to induce rat aortic ring contraction and to activate Gq and G 12 signaling pathways. We identified [Pep 6 ]URP, that binds UT with an affinity similar to that of URP, but behaves as a biased ligand. Used as an antagonist, this peptide is also able to selectively reduce the maximal aortic contraction of URP but not UII. Our results suggest that the orientation of the Tyr residue can stabilize at least two different conformations of UT, leading to biased signaling and a probe-dependent allosteric effect. [ABSTRACT FROM AUTHOR]

In addition to cell surface membranes, numerous G protein–coupled receptors (GPCRs) are located on intracellular membranes including the nuclear envelope. Although the role of numerous GPCRs at the cell surface has been well characterized, the physiological function of these same receptors located on intracellular membranes remains to be determined. Here, we employ a novel caged Ang-II analog, cAng-II, to compare the effects of the activation of cell surface versus intracellular angiotensin receptors in intact cardiomyocytes. When added extracellularly to HEK 293 cells, Ang-II and photolysed cAng-II increased ERK1/2 phosphorylation (via AT1R) and cGMP production (AT2R). In contrast unphotolysed cAng-II did not. Cellular uptake of cAng-II was 6-fold greater than that of Ang-II and comparable to the HIV TAT(48–60) peptide. Intracellular photolysis of cAng-II induced an increase in nucleoplasmic Ca 2+ ([Ca 2+ ] n ) that was greater than that induced by extracellular application of Ang-II. We conclude that cell-permeable ligands that can access intracellular GPCRs may evoke responses distinct from those with access restricted to the same receptor located on the cell surface. [ABSTRACT FROM AUTHOR]

Class B G protein-coupled receptors are activated by their cognate ligands following a two-step binding model involving a specific network of ligand-receptor intermolecular interactions. In particular, a N-capping structure present in the ligand would contribute significantly to position the N-terminal segment of the ligand once bound to its receptor. The aim of the current study was to implement the use of Pd-catalyzed Sonogashira coupling for the investigation of this structural motif. First, we have developed and evaluated various Sonogashira-based procedures for on-resin post-synthesis modification using a Leu-enkephalin derivative as a model peptide. Next, we have prepared a small library of PACAP-based analogs and evaluated the pharmacological profile of a few of them using a competitive binding assay, as well as functional and survival assays. Notably, our results suggest that the modification of the N-capping region could alter the binding specificity of PACAP without altering its biological activity, thereby opening the way for the design of more selective compounds. Finally, the possibility to achieve sequential multiple point substitutions via the Sonogashira cross-coupling method, during solid phase peptide synthesis, was also evaluated. Altogether, we demonstrated the versatility of such a procedure for the incorporation of various mono- and multiple alkyne-derived modifications during solid phase peptide synthesis and confirmed its usefulness for the structure-activity study of a class B GPCR ligand. [ABSTRACT FROM AUTHOR]

The pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1), a class B G protein-coupled receptor (GPCR), has emerged as a promising target for treating neurodegenerative conditions. Unfortunately, despite years of research, no PAC1-specific agonist has been discovered, as activity on two other GPCRs, VPAC1 and VPAC2, is retained with current analogs. Cell signaling is related to structural modifications in the intracellular loops (ICLs) of GPCRs. Thus, we hypothesized that peptides derived from the ICLs (called pepducins) of PAC1 might initiate, as allosteric ligands, signaling cascades after recognition of the parent receptor and modulation of its conformational landscape. Three pepducins were synthesized and evaluated for their ability to 1) promote cell survival; 2) stimulate various signaling pathways associated with PAC1 activation; 3) modulate selectively PAC1, VPAC1 or VPAC2 activation; and 4) sustain mobility and prevent death of dopaminergic neurons in a zebrafish model of neurodegeneration. Assays demonstrated that these molecules promote SH-SY5Y cell survival, a human neuroblastoma cell line expressing PAC1, and activate signaling via Gα s and Gα q , with distinct potencies and efficacies. Also, PAC1-Pep1 and PAC1-Pep2 activated selectively PAC1-mediated Gα s stimulation. Finally, experiments, using a zebrafish neurodegeneration model, showed a neuroprotective action with all three pepducins and in particular, revealed the ability of PAC1-Pep1 and PAC1-Pep3 to preserve fish mobility and tyrosine hydroxylase expression in the brain. We have developed the first neuroprotective pepducins derived from PAC1, a class B GPCR. PAC1-derived pepducins represent attractive templates for the development of innovative neuroprotecting molecules. • PAC1-derived pepducins can promote SH-SY5Y cell survival. • PAC1-Pep1 and PAC1-Pep2 can selectively trigger PAC1-mediated Gas stimulation. • PAC1-derived pepducins preserve fish mobility and tyrosine hydroxylase expression. [ABSTRACT FROM AUTHOR]