Evidence for heterodimerization and functional interaction of the urotensin II and the angiotensin II type 1 receptors.

Despite the observation of synergistic interactions between the urotensinergic and angiotensinergic systems, the interplay between the urotensin II receptor (hUT) and the angiotensin II type 1 receptor (hAT1R) in regulating cellular signaling remains incompletely understood. Notably, the putative interaction between hUT and hAT1R could engender reciprocal allosteric modulation of their signaling signatures, defining a unique role for these complexes in cardiovascular physiology and pathophysiology. Using a combination of co-immunoprecipitation, bioluminescence resonance energy transfer (BRET) and FlAsH BRET-based conformational biosensors, we first demonstrated the physical interaction between hUT and hAT1R. Next, to analyze how this functional interaction regulated proximal and distal hUT- and hAT1R-associated signaling pathways, we used BRET-based signaling biosensors and western blots to profile pathway-specific signaling in HEK 293 cells expressing hUT, hAT1R or both. We observed that hUT-hAT1R heterodimers triggered distinct signaling outcomes compared to their respective parent receptors alone. Notably, co-transfection of hUT and hAT1R has no impact on hUII-induced Gq activation but significantly reduced the potency and efficacy of Ang II to mediate Gq activation. Interestingly, URP, the second hUT endogenous ligand, produce a distinct signaling signature compared to hUII at hUT-hAT1R. Our results therefore suggest that assembly of hUT with hAT1R might be important for allosteric modulation of outcomes associated with specific hardwired signaling complexes in healthy and disease states. Altogether, our work, which potentially explains the interplay observed in native cells and tissues, validates such complexes as potential targets to promote the design of compounds that can modulate heterodimer function selectively.

Urotensin II Receptor Modulation with 1,3,4-Benzotriazepin-2-one Tetrapeptide Mimics.

Urotensin II receptor (UT) modulators that differentiate the effects of the endogenous cyclic peptide ligands urotensin II (UII) and urotensin II-related peptide (URP) offer potential for dissecting their respective biological roles in disease etiology. Selective modulators of hUII and URP activities were obtained using 1,3,4-benzotriazepin-2-one mimics of a purported bioactive γ-turn conformation about the Bip-Lys-Tyr tripeptide sequence of urocontrin ([Bip4]URP). Considering an active ß-turn conformer about the shared Phe-Trp-Lys-Tyr sequence of UII and URP, 8-substituted 1,3,4-benzotriazepin-2-ones were designed to mimic the Phe-Bip-Lys-Tyr tetrapeptide sequence of urocontrin, synthesized, and examined for biological activity. Subtle 5- and 8-position modifications resulted in biased signaling and selective modulation of hUII- or URP-induced vasoconstriction. For example, p-hydroxyphenethyl analogs 17b-d were strong Gα13 and ßarr1 activators devoid of Gαq-mediated signaling. Tertiary amides 15d and 17d negatively modulated hUII-induced vasoconstriction without affecting URP-mediated responses. Benzotriazepinone carboxamides proved to be exceptional tools for elucidating the pharmacological complexity of UT.

G Protein-Biased Agonists for Intracellular Angiotensin Receptors Promote Collagen Secretion in Myofibroblasts.

Photoactivatable ligands remain valuable tools to study the spatiotemporal aspects of cellular signaling. However, the synthesis, handling, and biological validation of such compounds remain challenging, especially when dealing with peptides. We report an optimized synthetic strategy, where laborious preparation of dimethoxy-nitrobenzyl-tyrosine building blocks was replaced by direct functionalization of amino acid side chains while peptides remained coupled to resin, reducing both preparation time and cost. Our caged peptides were designed to investigate cellular responses mediated by intracellular angiotensin II receptors (iATR) upon interaction with known biased and unbiased ligands. The pathophysiological roles of iATRs remain poorly understood, and we sought to develop ligands to explore this. Initial validation showed that our caged ligands undergo rapid photolysis and produced functionally active peptides upon UV exposure. We also show, for the first time, that different biased ligands (ß-arrestin- vs G protein-biased analogues) evoked distinct responses when uncaged in adult rat myofibroblasts. Intracellularly targeted versions of Ang II (unbiased) or G protein-biased analogues (TRV055, TRV056) were more effective than ß-arrestin-biased Ang II analogues (SI, TRV026, and TRV27) in inducing collagen secretion, suggesting a divergent role in regulating the fibrotic response.

Exploration of the urocontrin A scaffold yields new urotensinergic system allosteric modulator and competitive antagonists.

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 Gq biased ligand with an insurmountable antagonism in our aortic ring contraction assay.

Placental Galectins in Cancer: Why We Should Pay More Attention.

The first studies suggesting that abnormal expression of galectins is associated with cancer were published more than 30 years ago. Today, the role of galectins in cancer is relatively well established. We know that galectins play an active role in many types of cancer by regulating cell growth, conferring cell death resistance, or inducing local and systemic immunosuppression, allowing tumor cells to escape the host immune response. However, most of these studies have focused on very few galectins, most notably galectin-1 and galectin-3, and more recently, galectin-7 and galectin-9. Whether other galectins play a role in cancer remains unclear. This is particularly true for placental galectins, a subgroup that includes galectin-13, -14, and -16. The role of these galectins in placental development has been well described, and excellent reviews on their role during pregnancy have been published. At first sight, it was considered unlikely that placental galectins were involved in cancer. Yet, placentation and cancer progression share several cellular and molecular features, including cell invasion, immune tolerance and vascular remodeling. The development of new research tools and the concomitant increase in database repositories for high throughput gene expression data of normal and cancer tissues provide a new opportunity to examine the potential involvement of placental galectins in cancer. In this review, we discuss the possible roles of placental galectins in cancer progression and why they should be considered in cancer studies. We also address challenges associated with developing novel research tools to investigate their protumorigenic functions and design highly specific therapeutic drugs.

Tunable Surface Charge Enables the Electrostatic Adsorption-Controlled Release of Neuroprotective Peptides from a Hydrogel-Nanoparticle Drug Delivery System.

We exploit the electrostatic interactions between the positively charged neuroprotective peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), and negatively charged poly(lactic-co-glycolic acid) (PLGA) nanoparticles to control PACAP release from the surface of nanoparticles dispersed in a hyaluronan-methylcellulose (HAMC) hydrogel composite. PACAP is a promising therapeutic for the treatment of neurological disorders, yet it has been difficult to deliver in vivo. Herein, the PACAP release rate was tuned by manipulating peptide adsorption onto the surface of blank nanoparticles by modifying either nanoparticle loading in the hydrogel or nanoparticle surface charge. This peptide-nanoparticle interaction was controlled by the pH-responsive carboxylic acid end terminal groups of PLGA. We further validated this system with the controlled release of a novel stabilized PACAP analog: Ac-[Ala15, Ala20]PACAP-propylamide, which masks its recognition to peptidases in circulation. Both wild-type and stabilized PACAP released from the vehicle increased the production of neuroprotective Interleukin-6 from cultured primary astrocytes. Using computational fluid dynamics methods, PACAP release from the composite was predicted based on experimentally derived adsorption isotherms, which exhibited similar release profiles to experimental data. This versatile adsorption-based system was used to deliver PACAP locally to the brains of stroke-injured mice over a 10 day period in vivo, highlighting its effectiveness for the controlled release of PACAP to the central nervous system.

The complicated lives of GPCRs in cardiac fibroblasts.

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.

Quantifying Biomolecular Interactions Using Slow Mixing Mode (SLOMO) Nanoflow ESI-MS.

Electrospray ionization mass spectrometry (ESI-MS) is a powerful label-free assay for detecting noncovalent biomolecular complexes in vitro and is increasingly used to quantify binding thermochemistry. A common assumption made in ESI-MS affinity measurements is that the relative ion signals of free and bound species quantitatively reflect their relative concentrations in solution. However, this is valid only when the interacting species and their complexes have similar ESI-MS response factors (RFs). For many biomolecular complexes, such as protein-protein interactions, this condition is not satisfied. Existing strategies to correct for nonuniform RFs are generally incompatible with static nanoflow ESI (nanoESI) sources, which are typically used for biomolecular interaction studies, thereby significantly limiting the utility of ESI-MS. Here, we introduce slow mixing mode (SLOMO) nanoESI-MS, a direct technique that allows both the RF and affinity (K d) for a biomolecular interaction to be determined from a single measurement using static nanoESI. The approach relies on the continuous monitoring of interacting species and their complexes under nonhomogeneous solution conditions. Changes in ion signals of free and bound species as the system approaches or moves away from a steady-state condition allow the relative RFs of the free and bound species to be determined. Combining the relative RF and the relative abundances measured under equilibrium conditions enables the K d to be calculated. The reliability of SLOMO and its ease of use is demonstrated through affinity measurements performed on peptide-antibiotic, protease-protein inhibitor, and protein oligomerization systems. Finally, affinities measured for the binding of human and bacterial lectins to a nanobody, a viral glycoprotein, and glycolipids displayed within a model membrane highlight the tremendous power and versatility of SLOMO for accurately quantifying a wide range of biomolecular interactions important to human health and disease.

Perturbing dimer interactions and allosteric communication modulates the immunosuppressive activity of human galectin-7.

The design of allosteric modulators to control protein function is a key objective in drug discovery programs. Altering functionally essential allosteric residue networks provides unique protein family subtype specificity, minimizes unwanted off-target effects, and helps avert resistance acquisition typically plaguing drugs that target orthosteric sites. In this work, we used protein engineering and dimer interface mutations to positively and negatively modulate the immunosuppressive activity of the proapoptotic human galectin-7 (GAL-7). Using the PoPMuSiC and BeAtMuSiC algorithms, mutational sites and residue identity were computationally probed and predicted to either alter or stabilize the GAL-7 dimer interface. By designing a covalent disulfide bridge between protomers to control homodimer strength and stability, we demonstrate the importance of dimer interface perturbations on the allosteric network bridging the two opposite glycan-binding sites on GAL-7, resulting in control of induced apoptosis in Jurkat T cells. Molecular investigation of G16X GAL-7 variants using X-ray crystallography, biophysical, and computational characterization illuminates residues involved in dimer stability and allosteric communication, along with discrete long-range dynamic behaviors involving loops 1, 3, and 5. We show that perturbing the protein-protein interface between GAL-7 protomers can modulate its biological function, even when the overall structure and ligand-binding affinity remains unaltered. This study highlights new avenues for the design of galectin-specific modulators influencing both glycan-dependent and glycan-independent interactions.

Lipidated peptides derived from intracellular loops 2 and 3 of the urotensin II receptor act as biased allosteric ligands.

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 hUT-Pep2 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 hUT-derived 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.

Binding of a Soluble meso-Tetraarylporphyrin to Human Galectin-7 Induces Oligomerization and Modulates Its Pro-Apoptotic Activity.

The selective targeting of protein-protein interactions remains a significant determinant for the proper modulation and regulation of cell apoptosis. Prototypic galectins such as human galectin-7 (GAL-7) are characterized by their ability to form homodimers that control the molecular fate of a cell by mediating subtle yet critical glycan-dependent interactions between pro- and anti-apoptotic molecular partners. Altering the structural architecture of GAL-7 can therefore result in resistance to apoptosis in various human cancer cells, further illustrating its importance in cell survival. In this study, we used a combination of biophysical and cellular assays to illustrate that binding of a water-soluble meso-tetraarylporphyrin molecule to GAL-7 induces protein oligomerization and modulation of GAL-7-induced apoptosis in human Jurkat T cells. Our results suggest that the integrity of the GAL-7 homodimer architecture is essential for its molecular function, in addition to providing an interesting porphyrin binding modulator for controlling apoptosis in mammalian cells.

Insights into the Molecular Determinants Involved in Urocontrin and Urocontrin A Action.

In the past few years, we have identified two allosteric modulators of the urotensinergic system with probe-dependent action, termed Urocontrin (UC) and Urocontrin A (UCA). Such action is atypical and important since it will allow us to understand the specific function of the functionally selective cognate ligands of this system, namely urotensin II and urotensin II-related peptide. Delineating the molecular determinants involved in this particular behavior would represent an important step toward designing small molecules suitable for pharmacologic and/or therapeutic intervention. Hence, we undertook an exploratory research by replacing the Trp4 residue of URP with several para-substituted phenylalanine amino acids in order to get a grasp on the required nature, distance, and orientation of the side chain of this residue for allosteric modulatory action. We found that the position of the second aromatic group is crucial, and we identified two new allosteric modulators: [Trip4]URP and [Phe(pPy-4)4]URP with probe-dependent action.

Spt5 Phosphorylation and the Rtf1 Plus3 Domain Promote Rtf1 Function through Distinct Mechanisms.

Rtf1 is a conserved RNA polymerase II (RNAPII) elongation factor that promotes cotranscriptional histone modification, RNAPII transcript elongation, and mRNA processing. Rtf1 function requires the phosphorylation of Spt5, an essential RNAPII processivity factor. Spt5 is phosphorylated within its C-terminal domain (CTD) by cyclin-dependent kinase 9 (Cdk9), the catalytic component of positive transcription elongation factor b (P-TEFb). Rtf1 recognizes phosphorylated Spt5 (pSpt5) through its Plus3 domain. Since Spt5 is a unique target of Cdk9 and Rtf1 is the only known pSpt5-binding factor, the Plus3/pSpt5 interaction is thought to be a key Cdk9-dependent event regulating RNAPII elongation. Here, we dissect Rtf1 regulation by pSpt5 in the fission yeast Schizosaccharomyces pombe We demonstrate that the Plus3 domain of Rtf1 (Prf1 in S. pombe) and pSpt5 are functionally distinct and that they act in parallel to promote Prf1 function. This alternate Plus3 domain function involves an interface that overlaps the pSpt5-binding site and that can interact with single-stranded nucleic acid or with the polymerase-associated factor (PAF) complex in vitro We further show that the C-terminal region of Prf1, which also interacts with PAF, has a similar parallel function with pSpt5. Our results elucidate unexpected complexity underlying Cdk9-dependent pathways that regulate transcription elongation.

Towards Targeting the Urotensinergic System: Overview and Challenges.

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.

Design and biological assessment of membrane-tethering neuroprotective peptides derived from the pituitary adenylate cyclase-activating polypeptide type 1 receptor.

BACKGROUND: 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. METHODS: 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. RESULTS: Assays demonstrated that these molecules promote SH-SY5Y cell survival, a human neuroblastoma cell line expressing PAC1, and activate signaling via Gαs and Gαq, with distinct potencies and efficacies. Also, PAC1-Pep1 and PAC1-Pep2 activated selectively PAC1-mediated Gαs 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. CONCLUSION: We have developed the first neuroprotective pepducins derived from PAC1, a class B GPCR. GENERAL SIGNIFICANCE: PAC1-derived pepducins represent attractive templates for the development of innovative neuroprotecting molecules.

Targeting the PAC1 Receptor for Neurological and Metabolic Disorders.

The pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor (PAC1R, ADCYAP1R1) is a member of the vasoactive intestinal peptide (VIP)/secretin/glucagon family of G protein-coupled receptors (GPCRs). PAC1R has been shown to play crucial roles in the central and peripheral nervous systems. The activation of PAC1R initiates diverse downstream signal transduction pathways, including adenylyl cyclase, phospholipase C, MEK/ERK, and Akt pathways that regulate a number of physiological systems to maintain functional homeostasis. Accordingly, at times of tissue injury or insult, PACAP/PAC1R activation of these pathways can be trophic to blunt or delay apoptotic events and enhance cell survival. Enhancing PAC1R signaling under these conditions has the potential to mitigate cellular damages associated with cerebrovascular trauma (including stroke), neurodegeneration (such as Parkinson's and Alzheimer's disease), or peripheral organ insults. Conversely, maladaptive PACAP/PAC1R signaling has been implicated in a number of disorders, including stressrelated psychopathologies (i.e., depression, posttraumatic stress disorder, and related abnormalities), chronic pain and migraine, and metabolic diseases; abrogating PAC1R signaling under these pathological conditions represent opportunities for therapeutic intervention. Given the diverse PAC1R-mediated biological activities, the receptor has emerged as a relevant pharmaceutical target. In this review, we first describe the current knowledge regarding the molecular structure, dynamics, and function of PAC1R. Then, we discuss the roles of PACAP and PAC1R in the activation of a variety of signaling cascades related to the physiology and diseases of the nervous system. Lastly, we examine current drug design and development of peptides and small molecules targeting PAC1R based on a number of structure- activity relationship studies and key pharmacophore elements. At present, the rational design of PAC1R-selective peptide or small-molecule therapeutics is largely hindered by the lack of structural information regarding PAC1R activation mechanisms, the PACAP-PAC1R interface, and the core segments involved in receptor activation. Understanding the molecular basis governing the PACAP interactions with its different cognate receptors will undoubtedly provide a basis for the development and/or refinement of receptor-selective therapeutics.

Functional Selectivity Revealed by N-Methylation Scanning of Human Urotensin II and Related Peptides.

In accordance with their common but also divergent physiological actions, human urotensin II (1) and urotensin II-related peptide (2) could stabilize specific urotensin II receptor (UTR) conformations, thereby activating different signaling pathways, a feature referred to as biased agonism or functional selectivity. Sequential N-methylation of the amides in the conserved core sequence of 1, 2, and fragment U-II4-11 (3) shed light on structural requirements involved in their functional selectivity. Thus, 18 N-methylated UTR ligands were synthesized and their biological profiles evaluated using in vitro competition binding assays, ex vivo rat aortic ring bioassays and BRET-based biosensor experiments. Biological activity diverged from that of the parent structures contingent on the location of amide methylation, indicating relevant hydrogen-bond interactions for the function of the endogenous peptides. Conformational analysis of selected N-methyl analogs indicated the importance of specific amide residues of 2 for the distinct pharmacology relative to 1 and 3.

Antibacterial properties of the pituitary adenylate cyclase-activating polypeptide: A new human antimicrobial peptide.

The Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP), a polycationic, amphiphilic and helical neuropeptide, is well known for its neuroprotective actions and cell penetrating properties. In the present study, we evaluated the potent antibacterial property of PACAP38 and related analogs against various bacterial strains. Interestingly, PACAP38 and related analogs can inhibit the growth of various bacteria including Escherichia coli (JM109), Bacillus subtilis (PY79), and the pathogenic Burkholderia cenocepacia (J2315). Investigation of the mechanism of action suggested that a PACAP metabolite, identified as PACAP(9-38), might indeed be responsible for the observed PACAP38 antibacterial action. Surprisingly, PACAP(9-38), which does not induce haemolysis, exhibits an increased specificity toward Burkholderia cenocepacia J2315 compared to other tested bacteria. Finally, the predisposition of PACAP(9-38) to adopt a π-helix conformation rather than an α-helical conformation like PACAP38 could explain this gain in specificity. Overall, this study has revealed a new function for PACAP38 and related derivatives that can be added to its pleiotropic biological activities. This innovative study could therefore pave the way toward the development of new therapeutic agents against multiresistant bacteria, and more specifically the Burkholderia cenocepacia complex.

Discovery of New Allosteric Modulators of the Urotensinergic System through Substitution of the Urotensin II-Related Peptide (URP) Phenylalanine Residue.

Urotensin II (UII) and urotensin II-related peptide (URP) are functionally selective, suggesting that these two hormones might play distinct physiological role through different interactions with their cognate receptor UT. Hypothesizing that the Phe3 residue of URP, which is also present in UII, is a key-element of its specific UT activation, we evaluated the impact of its replacement by non-natural amino acids in URP. Each compound was evaluated for its ability to bind UT, induce rat aortic ring contraction, and activate Gq, G12, and ß-arrestin 1 signaling pathways. Such modifications impaired contractile efficacy, reflected by a reduced aptitude to activate G12 in URP but not in the truncated but equipotent UII4-11. Moreover, we have identified two structurally different UT modulators: [d-Phe(pI)3]URP and [Bip3]URP, which exert a probe-dependent action against UII and URP. These compounds should help us understand the specific roles of these hormones as well as guide further therapeutic development.

New insights about the peculiar role of the 28-38 C-terminal segment and some selected residues in PACAP for signaling and neuroprotection.

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 Gs and Gq, as well as ß-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 Gq and Gs-dependent signaling. This study will definitely improve our understanding of the molecular and cellular mechanisms associated with PACAP neuroprotection.