Therapeutic Potential of Targeting Prokineticin Receptors in Diseases.

The prokineticins (PKs) were discovered approximately 20 years ago as small peptides inducing gut contractility. Today, they are established as angiogenic, anorectic, and proinflammatory cytokines, chemokines, hormones, and neuropeptides involved in variety of physiologic and pathophysiological pathways. Their altered expression or mutations implicated in several diseases make them a potential biomarker. Their G-protein coupled receptors, PKR1 and PKR2, have divergent roles that can be therapeutic target for treatment of cardiovascular, metabolic, and neural diseases as well as pain and cancer. This article reviews and summarizes our current knowledge of PK family functions from development of heart and brain to regulation of homeostasis in health and diseases. Finally, the review summarizes the established roles of the endogenous peptides, synthetic peptides and the selective ligands of PKR1 and PKR2, and nonpeptide orthostatic and allosteric modulator of the receptors in preclinical disease models. The present review emphasizes the ambiguous aspects and gaps in our knowledge of functions of PKR ligands and elucidates future perspectives for PK research. SIGNIFICANCE STATEMENT: This review provides an in-depth view of the prokineticin family and PK receptors that can be active without their endogenous ligand and exhibits "constitutive" activity in diseases. Their non- peptide ligands display promising effects in several preclinical disease models. PKs can be the diagnostic biomarker of several diseases. A thorough understanding of the role of prokineticin family and their receptor types in health and diseases is critical to develop novel therapeutic strategies with safety concerns.

MRAP2 Inhibits ß-Arrestin-2 Recruitment to the Prokineticin Receptor 2.

Melanocortin receptor accessory protein 2 (MRAP2) is a membrane protein that binds multiple G protein-coupled receptors (GPCRs) involved in the control of energy homeostasis, including prokineticin receptors. These GPCRs are expressed both centrally and peripherally, and their endogenous ligands are prokineticin 1 (PK1) and prokineticin 2 (PK2). PKRs couple all G-protein subtypes, such as Gαq/11, Gαs, and Gαi, and recruit ß-arrestins upon PK2 stimulation, although the interaction between PKR2 and ß-arrestins does not trigger receptor internalisation. MRAP2 inhibits the anorexigenic effect of PK2 by binding PKR1 and PKR2. The aim of this work was to elucidate the role of MRAP2 in modulating PKR2-induced ß-arrestin-2 recruitment and ß-arrestin-mediated signalling. This study could allow the identification of new specific targets for potential new drugs useful for the treatment of the various pathologies correlated with prokineticin, in particular, obesity.

MRAP2a Binds and Modulates Activity and Localisation of Prokineticin Receptor 1 in Zebrafish.

The prokineticin system plays a role in hypothalamic neurons in the control of energy homeostasis. Prokineticin receptors (PKR1 and PKR2), like other G-protein-coupled receptors (GPCRs) are involved in the regulation of energy intake and expenditure and are modulated by the accessory membrane protein 2 of the melanocortin receptor (MRAP2). The aim of this work is to characterise the interaction and regulation of the non-melanocortin receptor PKR1 by MRAP2a in zebrafish (zMRAP2a) in order to use zebrafish as a model for the development of drugs targeting accessory proteins that can alter the localisation and activity of GPCRs. To this end, we first showed that zebrafish PKR1 (zPKR1) is able to interact with both zMRAP2a and human MRAP2 (hMRAP2). This interaction occurs between the N-terminal region of zPKR1 and the C-terminal domain of zMRAP2a, which shows high sequence identity with hMRAP2 and a similar propensity for dimer formation. Moreover, we demonstrated that in Chinese hamster ovary (CHO) cells, zMRAP2a or hMRAP2 are able to modulate zPKR1 activation induced by zebrafish PK2 (zPK2) resulting in an impaired ERK and STAT3 activation.

Special Issue "GPCR: Roles in Cell Development and Disease".

We are pleased to present the following Special Issue of the International Journal of Molecular Sciences (IJMS), entitled "GPCR: Roles in Cell Development and Disease" [...].

Genetic Incorporation of Dansylalanine in Human Ferroportin to Probe the Alternating Access Mechanism of Iron Transport.

Ferroportin (Fpn), a member of the major facilitator superfamily (MFS) of transporters, is the only known iron exporter found in mammals and plays a crucial role in regulating cellular and systemic iron levels. MFSs take on different conformational states during the transport cycle: inward open, occluded, and outward open. However, the precise molecular mechanism of iron translocation by Fpn remains unclear, with conflicting data proposing different models. In this work, amber codon suppression was employed to introduce dansylalanine (DA), an environment-sensitive fluorescent amino acid, into specific positions of human Fpn (V46, Y54, V161, Y331) predicted to undergo major conformational changes during metal translocation. The results obtained indicate that different mutants exhibit distinct fluorescence spectra depending on the position of the fluorophore within the Fpn structure, suggesting that different local environments can be probed. Cobalt titration experiments revealed fluorescence quenching and blue-shifts of λmax in Y54DA, V161DA, and Y331DA, while V46DA exhibited increased fluorescence and blue-shift of λmax. These observations suggest metal-induced conformational transitions, interpreted in terms of shifts from an outward-open to an occluded conformation. Our study highlights the potential of genetically incorporating DA into Fpn, enabling the investigation of conformational changes using fluorescence spectroscopy. This approach holds great promise for the study of the alternating access mechanism of Fpn and advancing our understanding of the molecular basis of iron transport.

Non-Peptide Agonists and Antagonists of the Prokineticin Receptors.

The prokineticin family comprises a group of secreted peptides that can be classified as chemokines based on their structural features and chemotactic and immunomodulatory functions. Prokineticins (PKs) bind with high affinity to two G protein-coupled receptors (GPCRs). Prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2) are involved in a variety of physiological functions such as angiogenesis and neurogenesis, hematopoiesis, the control of hypothalamic hormone secretion, the regulation of circadian rhythm and the modulation of complex behaviors such as feeding and drinking. Dysregulation of the system leads to an inflammatory process that is the substrate for many pathological conditions such as cancer, pain, neuroinflammation and neurodegenerative diseases such as Alzheimer's and Parkinson's disease. The use of PKR's antagonists reduces PK2/PKRs upregulation triggered by various inflammatory processes, suggesting that a pharmacological blockade of PKRs may be a successful strategy to treat inflammatory/neuroinflammatory diseases, at least in rodents. Under certain circumstances, the PK system exhibits protective/neuroprotective effects, so PKR agonists have also been developed to modulate the prokineticin system.

Arginine 125 Is an Essential Residue for the Function of MRAP2.

MRAP2 is a small simple transmembrane protein arranged in a double antiparallel topology on the plasma membrane. It is expressed in the paraventricular nucleus of the hypothalamus, where it interacts with various G protein-coupled receptors, such as the prokineticin receptors, and regulates energy expenditure and appetite. The aim of this work was to analyze the functional role of the specific arginine residue at position 125 of MRAP2, which affects protein conformation, dimer formation, and PKR2 binding. Results obtained with the MRAP2 mutants R125H and R125C, which are found in human patients with extreme obesity, and mouse MRAP2, in which arginine 125 is normally replaced by histidine, were compared with those obtained with human MRAP2. Understanding the mechanism by which MRAP2 regulates G protein-coupled receptors helps in elucidating the metabolic pathways involved in metabolic dysfunction and in developing new drugs as specific targets of the MRAP2-PKR2 complex.

Trypanosoma cruzi trans-sialidase induces STAT3 and ERK activation by prokineticin receptor 2 binding.

Tc85, as other members of trans-sialidase family, is involved in Trypanosoma cruzi parasite adhesion to mammalian cells. Particularly, Tc85 acts through specific interactions with prokineticin receptor 2, a G-protein coupled receptor involved in diverse physiological and pathological processes. In this manuscript, through biochemical analyses, we demonstrated that LamG, a Tc85 domain, physically interacts with the prokineticin receptor 2. Moreover, expressing prokineticin receptor 1 and 2 we demonstrated that LamG specifically activates prokineticin receptor 2 through a strong coupling with Gαi or Gαq proteins in yeast strains and inducing ERK and NFAT phosphorylation in CHO mammalian cells. To demonstrate a Tc85 physiological role in T. cruzi infection of the nervous system, we evidenced a strong STAT3 and ERK activation by LamG in mice Dorsal Root Ganglia. L173R is the most common prokineticin receptor 2 mutation reported in Kallmann syndrome and it is a founder mutation. Our results demonstrated that in cells co-expressing prokineticin receptor 2 mutant (L173R) and wild-type, LamG is unable to induce signal transduction. The L173R mutation in heterozygosity may allow for a selective advantage due to increased protection from T. cruzi infection. SIGNIFICANCE OF THE STUDY: The Chagas' disease affecting millions of people worldwide is caused by an eukaryotic microorganism called T. cruzi. Pharmacological treatment for patients with Chagas' disease is still limited. Indeed, the small number of drugs available shows important side effects that can be debilitating for patient health. In order to replicate and produce new parasites T. cruzi uses a complex of different proteins produced by both the parasite and the human host cells. So, understanding the molecular details used by T. cruzi to be internalised by different types of human cells is an important step towards the development of new drugs for this disease. Prokineticin receptors are relevant for host-parasite interaction. To characterise the signal transduction cascade induced by their activation may help to understand the molecular details of cell infection, leading to novel therapeutic alternative for this debilitating disease.

Biochemical characterization of Prokineticin 2 binding to Prokineticin receptor 1 in zebrafish.

Prokineticin 2 (PK2) binds to prokineticin receptor 1 and prokineticin receptor 2 (PKR1 and PKR2, respectively), two G protein-coupled receptors (GPCRs) that can mediate multiple signalling pathways by promoting the elevation of intracellular calcium and cAMP levels, phosphorylation of Akt and activation of ERK and STAT3. This work aims to evidence the conservation of protein sequence and the mechanism of PK2 binding to PKR1 to use the zebrafish model for the identification of new drugs as targets of prokineticin receptors. To this end, we first demonstrated that the zebrafish genes pk2 and pkr1 are phylogenetically related to orthologous mammalian genes by constructing evolutionary trees and performing syntenic analyses. Subsequently, by comparing the amino acid sequences, we showed that the interaction sites with PK2 are conserved in the zPKR1. Using GST pull-down and cross-linking experiments, we demonstrated the crucial role of the N-terminal region of zPKR1 for binding to the PK2. Finally, by expressing zPKR1 in CHO cells, we demonstrated the ability of zPKR1 to induce the activation of ERK and STAT3.

Special Issue "G Protein-Coupled Receptors: Molecular Mechanisms Involved in Receptor Activation and Selectivity".

Welcome to the Special Issue of Life entitled "G Protein-Coupled Receptors: Molecular Mechanisms in Receptor Activation and Selectivity" [...].

Blocking prokineticin receptors attenuates synovitis and joint destruction in collagen-induced arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease mediated by an interdependent network of proinflammatory molecules such as chemokines. Prokineticin 2 (PK2) is a chemokine-like peptide that modulates nociceptive threshold and immuno-inflammatory processes via two G-protein-linked receptors, prokineticin receptor 1 and 2 (PKR1 and PKR2). In the present study, we investigated the effects of the prokineticin receptor antagonist PC1 on arthritic pain and the inflammatory response in type II collagen-induced arthritis (CIA) in mice. We demonstrated that PC1, administered subcutaneously from day 25 to day 35 after CIA, improved clinical signs of arthritis such as paw edema, pain, and impaired locomotor activity. In CIA mice, PC1 was also able to lower plasma malondialdehyde (MDA) levels, suggesting a role in reducing oxidative damage, as well as joint expression levels of PK2, PKRs, TNFα, IL-1ß, CD4, CD8, and NF-kB. These results suggest that blocking PKRs may be a successful strategy to control arthritic pain and pathology development. KEY MESSAGES: PK2/PKRs expression levels strongly increase in the synovium of RA mice. PC1 treatment shows anti-arthritic activity and reduces arthritis-induced pain. PC1 treatment significantly lowers synovial PK2/PKRs levels. PC1 treatment lowers plasma MDA levels and synovial levels of TNFα and IL -1ß PC1 treatment is a viable therapeutic option for RA.

Evidence that prokineticin receptor 2 exists as a dimer in vivo.

Prokineticins are proteins that regulate diverse biological processes including gastrointestinal motility, angiogenesis, circadian rhythm, and innate immune response. Prokineticins bind two closed related G-protein coupled receptors (GPCRs), PKR1 and PKR2. In general, these receptors act as molecular switches to relay activation to heterotrimeric G-proteins and a growing body of evidence points to the fact that GPCRs exist as homo- or heterodimers. We show here by Western-blot analysis that PKR2 has a dimeric structure in neutrophils. By heterologous expression of PKR2 in Saccharomyces cerevisiae, we examined the mechanisms of intermolecular interaction of PKR2 dimerization. The potential involvement of three types of mechanisms was investigated: coiled-coil, disulfide bridges, and hydrophobic interactions between transmembrane domains. Characterization of differently deleted or site-directed PKR2 mutants suggests that dimerization proceeds through interactions between transmembrane domains. We demonstrate that co-expressing binding-deficient and signaling-deficient forms of PKR2 can re-establish receptor functionality, possibly through a domain-swapping mechanism.

Prokineticin-Receptor Network: Mechanisms of Regulation.

Prokineticins are a new class of chemokine-like peptides that bind their G protein-coupled receptors, PKR1 and PKR2, and promote chemotaxis and the production of pro-inflammatory cytokines following tissue injury or infection. This review summarizes the major cellular and biochemical mechanisms of prokineticins pathway regulation that, like other chemokines, include: genetic polymorphisms; mRNA splice modulation; expression regulation at transcriptional and post-transcriptional levels; prokineticins interactions with cell-surface glycosaminoglycans; PKRs degradation, localization, post-translational modifications and oligomerization; alternative signaling responses; binding to pharmacological inhibitors. Understanding these mechanisms, which together exert substantial biochemical control and greatly enhance the complexity of the prokineticin-receptor network, leads to novel opportunities for therapeutic intervention. In this way, besides targeting prokineticins or their receptors directly, it could be possible to indirectly influence their activity by modulating their expression and localization or blocking the downstream signaling pathways.

Prokineticin 2 in cancer-related inflammation.

Inflammation, which triggers the release of a variety of growth factors, cytokines, and chemokines, is a critical component of tumor progression. Prokineticin 2 belongs to a new family of chemokines bound to two G-protein-coupled receptors called prokineticin receptor 1 and 2 that exert various tissue-specific biological functions. Under pathological conditions, prokineticin 2 can induce the proliferation, migration, and angiogenesis of endothelial cells, suggesting that this molecule plays a role in tumor growth, angiogenesis, and metastasis. The aim of this review is to provide a complete compendium of the involvement of prokineticin 2 in some cancers and to evaluate its role not only in the tumor microenvironment as an angiogenic factor and a mediator of immune cell migration, but also in modulating tumor growth and spread as a suppressor of tumor cell apoptosis, and as a trigger of their proliferation and movements required for metastasis. The involvement of prokineticin 2 in tumor pain and resistance responses is also described, and finally, the potential role of prokineticin 2 as a novel prognostic tumor biomarker is highlighted.

Identification and Characterization of a New Splicing Variant of Prokineticin 2.

Prokineticin 2 (PROK2) is a secreted bioactive peptide that regulates a variety of biological responses via two GPCRs, the prokineticin receptors (PROKRs). The aim of this study was to characterize a new alternatively spliced product of the prok2 gene consisting of four exons. The 40-amino acid peptide, designated PROK2C, is encoded by exon 1 and exon 4, and its expression was detected in the hippocampus and spinal cord of mice. PROK2C was expressed in a heterologous system, Pichia pastoris, and its binding specificity to the amino-terminal regions of PROKR1 and PROKR2 was investigated by GST pull-down experiments. In addition, the introduction of the unnatural amino acid p-benzoyl-L-phenylalanine using amber codon suppression technology demonstrated the role of tryptophan at position 212 of PROKR2 for PROK2C binding by photoactivatable cross-linking. The functional significance of this new isoform was determined in vivo by nociceptive experiments, which showed that PROK2C elicits strong sensitization of peripheral nociceptors to painful stimuli. In order to analyze the induction of PROK2C signal transduction, STAT3 and ERK phosphorylation levels were determined in mammalian CHO cells expressing PROKR1 and PROKR2. Our data show by in vivo and in vitro experiments that PROK2C can bind and activate both prokineticin receptors.

Identification of Regions Involved in the Physical Interaction between Melanocortin Receptor Accessory Protein 2 and Prokineticin Receptor 2.

Melanocortin Receptor Accessory Protein 2 (MRAP2) modulates the trafficking and signal transduction of several G-protein-coupled receptors (GPCRs) involved in the control of energy homeostasis, such as Prokineticin receptors (PKRs). They bind the endogenous ligand prokineticin 2 (PK2), a novel adipokine that has an anorexic effect and modulates thermoregulation and energy homeostasis. In the present work, we used biochemical techniques to analyze the mechanism of interaction of MRAP2 with PKR2 and we identified the specific amino acid regions involved in the complex formation. Our results indicate that MRAP2 likely binds to the N-terminal region of PKR2, preventing glycosylation and consequently the correct receptor localization. We also identified a C-terminal region of MRAP2 that is critical for the interaction with PKR2. Consequently, we analyzed the role of the prokineticin transduction system in the regulation of MRAP2 expression in tissues involved in the control of food intake: at the central level, in hypothalamic explants, and at the peripheral level, in adipocytes. We demonstrated the modulation of MRAP2 expression by the prokineticin transduction system.

Versatile Role of Prokineticins and Prokineticin Receptors in Neuroinflammation.

Prokineticins are a new class of chemokine-like peptides involved in a wide range of biological and pathological activities. In particular, prokineticin 2 (PK2), prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2) play a central role in modulating neuroinflammatory processes. PK2 and PKRs, which are physiologically expressed at very low levels, are strongly upregulated during inflammation and regulate neuronal-glial interaction. PKR2 is mainly overexpressed in neurons, whereas PKR1 and PK2 are mainly overexpressed in astrocytes. Once PK2 is released in inflamed tissue, it is involved in both innate and adaptive responses: it triggers macrophage recruitment, production of pro-inflammatory cytokines, and reduction of anti-inflammatory cytokines. Moreover, it modulates the function of T cells through the activation of PKR1 and directs them towards a pro-inflammatory Th1 phenotype. Since the prokineticin system appears to be upregulated following a series of pathological insults leading to neuroinflammation, we will focus here on the involvement of PK2 and PKRs in those pathologies that have a strong underlying inflammatory component, such as: inflammatory and neuropathic pain, Alzheimer's disease, Parkinson's disease, multiple sclerosis, stroke, obesity, diabetes, and gastrointestinal inflammation.

The balance of concentration between Prokineticin 2ß and Prokineticin 2 modulates the food intake by STAT3 signaling.

The secreted bioactive peptide prokineticin 2 (PK2) is a potent adipokine and its central and peripheral administration reduces food intake in rodents. The pk2 gene has two splice variants, PK2 and PK2L (PK2 long form), which is cleaved into an active peptide, PK2ß, that preferentially binds prokineticin receptor 1 (PKR1). We investigated the role of PK2ß in the regulation of food intake. We demonstrated that intraperitoneal injection of PK2ß, in contrast to PK2, did not reduce food intake in mice. Exposure of hypotalamic explants to PK2, but not PK2ß, induced phosphorylation of STAT3 and ERK. We also evidenced that in adipocytes from PKR1 knock-out mice, a model of obesity, there were higher PK2ß levels than PK2 inducing a decreased activation of STAT3 and ERK. Our results suggest that variations in PK2 and PK2ß levels, due to modulation of pk2 gene splicing processes, affect food intake in mice.

Analysis of role of aromatic residues in extracellular loop 2 of Prokineticin receptor 2 in ligand binding probed with genetically encoded photo-crosslinkers.

Prokineticin 2 (PK2) and Prokineticin 2 beta (PK2ß), products of alternative splicing of pk2 gene, are chemokine-like proteins. While PK2 mediates its biological activities by signaling with the same efficiency through two homologous G protein coupled receptors, prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2), PK2ß is able to bind specifically PKR1. Extracellular loop 2 (ECL2) of chemokine receptors is a part of a transmembrane (TM) ligand binding site. In the ECL2 of PKR2 is present, as well as in all chemokine receptors, an aromatic residue cluster, involving tryptophan 212 localized four residues after an ECL2 conserved cysteine, and Phenylalanine 198 located in the top of TM 4. In this work, the photoactivatable unnatural amino acid p-benzoyl-L-phenylalanine is incorporated by amber codon suppression technology into PKR2 in position 212. Experiments of photoactivatable cross-linking demonstrated the role of tryptophan in position 212 for binding the ligand contacting Tryptophan in position 24. We also analyzed the role of Phenylalanine 198 in the specificity of PKRs binding. The comparison of TM-bundle binding sites between PKR1 and PKR2 revealed that they are completely conserved except for one residue: valine 207 in human PKR1, which is phenylalanine 198 in human PKR2. The F198V mutation in PKR2 permits to obtain a receptor able to bind more efficiently PK2ß, a ligand highly specific for PKR1.

Expression of Bv8 in Pichia pastoris to identify structural features for receptor binding.

Bv8 is an amphibian peptide belonging to the widely distributed AVIT protein family. The mammalian orthologues of Bv8 were named prokineticin 1 and prokineticin 2. Two G-protein-coupled receptors for Bv8-prokineticins have been identified. The biological activities of Bv8/PK proteins range from angiogenesis and involvement in reproduction and cancer, to neuronal survival and neurogenesis, hypothalamic hormone secretion, circadian rhythm control and immunomodulatory processes. Identifying the structural determinants required for receptor binding of Bv8-PKs is mandatory for the design of PKR antagonists, which may be useful in the treatment and prevention of various disease states. Here we describe a procedure for the production in Pichia pastoris of Bv8 and 3 mutants: W24A-Bv8, in which the tryptophan in position 24 is substituted by alanine, the double mutant M1-W24A-Bv8, that contains an additional methionine at the N-terminus and Bv8-TyrTyr that includes two additional tyrosines at the C-terminus. The results evidence a relevant role of tryptophan 24 in Bv8-PKRs interaction.