Prokineticins and their G protein-coupled receptors in health and disease.

Prokineticins are two conserved small proteins (~8kDa), prokineticin 1 (PROK1; also called EG-VEGF) and prokineticin 2 (PROK2; also called Bv8), with an N-terminal AVITGA sequence and 10 cysteines forming 5 disulfide bridges. PROK1 and PROK2 bind to two highly related G protein-coupled receptors (GPCRs), prokineticin receptor 1 (PROKR1) and prokineticin receptor 2 (PROKR2). Prokineticins and their receptors are widely expressed. PROK1 is predominantly expressed in peripheral tissues, especially steroidogenic organs, whereas PROK2 is mainly expressed in the central nervous system and nonsteroidogenic cells of the testes. Prokineticins signaling has been implicated in several important physiological functions, including gastrointestinal smooth muscle contraction, circadian rhythm regulation, neurogenesis, angiogenesis, pain perception, mood regulation, and reproduction. Dysregulation of prokineticins signaling has been observed in a variety of diseases, such as cancer, ischemia, and neurodegeneration, in which prokineticins signaling seems to be a promising therapeutic target. Based on the phenotypes of knockout mice, PROKR2 and PROK2 have recently been identified as causative genes for idiopathic hypogonadotropic hypogonadism, a developmental disorder characterized by impaired development of gonadotropin-releasing hormone neurons and infertility. In vitro functional studies with these disease-associated PROKR2 mutations uncovered some novel features for this receptor, such as biased signaling, which may be used to understand GPCR signaling regulation in general.

A Prokineticin (PK)-like cytokine from Chinese mitten crab Eriocheir sinensis promotes the production of hemocytes via reactive oxygen species.

Astakine is a cytokine-like factor containing a prokineticin domain, which directly participates in hematopoiesis and blood cell differentiation. In the present study, a novel Astakine gene was identified from Chinese mitten crab Eriocheir sinensis (designated as EsAst). The full-length cDNA of EsAst was of 1163 bp, consisting of a 5' untranslated region (UTR) of 120 bp, a 3' UTR of 656 bp, and an open reading frame (ORF) of 387 bp encoding a polypeptide of 128 amino acids. There were a signal peptide and a prokineticin domain with nine conserved cysteine residues in the deduced amino acid sequence of EsAst. EsAst shared higher similarity with Astakines from Penaeus monodon and Pacifastacus leniusculus, and it was closely clustered with the Astakine from shrimp P. monodon in the phylogenetic tree. The EsAst mRNA transcript was higher expressed in hemocytes and hepatopancreas. The relative expression level of EsAst in hemocytes was continuously increased from 1.5 to 48 h after Vibro anguillarum challenge compared that in the untreated control group. After Pichia pastoris GS115 challenge, the relative expression level of EsAst in hemocytes was also up-regulated. After rEsAst injection, ROS levels in HPT cells were also increased at 12 and 24 h, and the total hemocyte counts were also significantly increased at 6, 9, 12, and 24 h post rEsAst injection. The interference of EsAst expression with dsRNA injection could delay the recovery of hemocytes production post A. hydrophila stimulation. When mitochondrial complexes I was knock down by dsRNA, ROS levels were decreased and THCs were also decreased. Recovery of hemocyte production inducing by A. hydrophila stimulation and rEsAst injection were delayed with dsEsbc1 injection. When ROS levels were increased after RNAi of Lon protease, THCs were also increased. The expression levels of five genes (EsJNK, EsSTAT, EsPI3K, EsAKT1, EsP70S6K) involved in SAPK-JNK and mTOR signaling pathways were up-regulated at 12 and 24 h in rEsAst group and EsLon dsRNA group compared with that in EGFP dsRNA group, and were similar to the trend of ROS levels. These results collectively suggested that EsAst should be a novel Astakine to promote the production of hemocytes in a ROS-dependent way in E. sinensis.

Targeting the Prokineticin System to Control Chronic Pain and Inflammation.

Prokineticin1 and prokineticin2 belong to a new family of chemokines identified in several species including mammals and characterized by the presence of five disulfide bridges. These proteins signal through two G-coupled receptors (prokineticin-receptor1 and prokineticin- receptor2) widely expressed in all tissues and involved in a large spectrum of biological activities, including angiogenesis, hematopoiesis, immune processes, inflammation and nociceptive transmission. Prokineticin2 is overexpressed in inflamed tissues and has a crucial role in neutrophil dependent inflammation and hypernociception. Following tissue inflammation, peripheral nerve injury, cancer, bone metastasis the expression of prokineticin2 and of the prokineticin-receptor2 is increased also within dorsal root ganglia and spinal cord. Prokineticin receptors, highly expressed in nociceptor endings and dorsal root ganglia, exert a tonic activation of TRPV1 and TRPA1 contributing to peripheral sensitization. Prokineticin2-induces activation of the prokineticin receptors in the spinal dorsal horn and in activated astrocytes contributes to central sensitization and maintains chronic and neuropathic pain. Prokineticin2, acting on prokineticin receptors on monocytes, macrophages and dendritic cells, induces chemotaxis and release of inflammatory and pronociceptive cytokines. Hence, the prokineticin system represents a novel therapeutic target in chronic pain conditions. Evaluation of the mechanism of action of prokineticin2 and the potential effectiveness of its inhibition is discussed.

Prokineticins in central and peripheral control of human reproduction.

Prokineticin 1 (PROK1) and (PROK2), are two closely related proteins that were identified as the mammalian homologs of their two amphibian homologs, mamba intestinal toxin (MIT-1) and Bv8. PROKs activate two G-protein linked receptors (prokineticin receptor 1 and 2, PROKR1 and PROKR2). Both PROK1 and PROK2 have been found to regulate a stunning array of biological functions. In particular, PROKs stimulate gastrointestinal motility, thus accounting for their family name "prokineticins". PROK1 acts as a potent angiogenic mitogen, thus earning its other name, endocrine gland-derived vascular endothelial factor. In contrast, PROK2 signaling pathway has been shown to be a critical regulator of olfactory bulb morphogenesis and sexual maturation. During the last decade, strong evidences established the key roles of prokineticins in the control of human central and peripheral reproductive processes. PROKs act as main regulators of the physiological functions of the ovary, uterus, placenta, and testis, with marked dysfunctions in various pathological conditions such as recurrent pregnancy loss, and preeclampsia. PROKs have also been associated to the tumor development of some of these organs. In the central system, prokineticins control the migration of GnRH neurons, a key process that controls reproductive functions. Importantly, mutations in PROK2 and PROKR2 are associated to the development of Kallmann syndrome, with direct consequences on the reproductive system. This review describes the finely tuned actions of prokineticins in the control of the central and peripheral reproductive processes. Also, it discusses future research directions for the use of these cytokines as diagnostic markers for several reproductive diseases.

Disease-causing mutation in PKR2 receptor reveals a critical role of positive charges in the second intracellular loop for G-protein coupling and receptor trafficking.

Prokineticins are a pair of signal factors involved in many physiological processes by binding to two closely related G-protein-coupled receptors, PKR1 and PKR2. Recently, mutations in prokineticin 2 (PK2) and PKR2 are found to be associated with Kallmann syndrome and/or idiopathic hypogonadotropic hypogonadism, disorders characterized by delayed puberty and infertility. However, little is known how PKRs interact and activate G-proteins to elicit signal transduction. In the present study, we took advantage of one disease-associated mutation (R164Q) located in the second intracellular (IL2) loop of PKR2, to investigate the role of IL2 loop in the cell signaling, G-protein binding and receptor trafficking. R164Q mutant PKR2 showed normal cell surface expression and ligand binding capacity. However, the PKR2 signaling was abolished by R164Q mutation. We demonstrated that R164Q mutation disrupted the interaction of IL2 loop to the Gα(q), Gα(i), and Gα(16)-proteins. A positive-charged amino acid at this position is required for proper function, and the signaling efficacy and potency depend on the net amount of positive charges. We also demonstrated that the interactive partner of Arg-164 may localize in the C-terminal five residues of Gα(q)-protein. A series of mutation analysis indicated that the basic amino acids at the C terminus of IL2 loop may function cooperatively in GPCRs. Furthermore, R164Q mutation also results in minimal ligand-induced endocytosis of PKR2. As many GPCRs share structural homology in the C terminus of IL2 loop, our findings may have general application in understanding structure and function of GPCRs.

Chemical synthesis and structure of the prokineticin Bv8.

Bv8, a 77-residue protein isolated from frogs, is the prototypic member of the prokineticin family of cytokines. Prokineticins (PKs) have only recently been identified in vertebrates (including humans), and they are believed to be involved in a number of key physiological processes, such as angiogenesis, neurogenesis, nociception, and tissue development. We used a combination of Boc solid-phase peptide synthesis, native chemical ligation, and in vitro protein folding to establish robust chemical access to this molecule. Synthetic Bv8 was obtained in good yield and exhibited full activity in a human neuroblastoma cell line and rat dorsal root ganglion (DRG) neurons. The 3D structure of the synthetic protein was determined by using NMR spectroscopy and it was found to be homologous with that of mamba intestinal toxin 1, which is the only other known prokineticin structure. Analysis of a truncated mutant lacking five residues at the N terminus that are critical for receptor binding and activation showed no perturbation to the core protein structure. Together with the functional data, this suggests that receptor binding is likely to be a highly cooperative process possibly involving major allosterically driven structural rearrangements. The facile and efficient synthesis presented here will enable preparation of unique chemical analogues of prokineticins, which should be powerful tools for modulating the structure and function of prokineticins and their receptors, and studying the many physiological processes that have been linked to them.

Cytokine properties of prokineticins.

Prokineticins are a novel family of secreted peptides with diverse regulatory roles, one of which is their capacity to modulate immunity in humans and in other species. Prokineticins are small peptides of 8 kDa that mediate their biological activities by signaling through two homologous G-protein-coupled receptors (prokineticin receptor 1 and prokineticin receptor 2). This family of peptides is characterized by a completely conserved N-terminal hexapeptide crucial for their bioactivities and a unique structural motif comprising five disulfide bonds. Prokineticins and their receptors are highly expressed in bone marrow, in peripheral circulating leukocytes, in inflamed tissues and in resident organ immune cells. Their structure, size, signaling and biological activities are reminiscent of the chemokine superfamily. In this review, emphasis is placed on the properties of prokineticins as cytokines and their role in the immune system.

Biological function of prokineticins.

Secreted peptides have been implicated in diverse physiological functions. Prokineticins are a pair of regulatory peptides that signal through two highly homologous G protein-coupled receptors. Prokineticins possess a unique structural motif of five disulfide bonds and conserved N-terminal stretches. Diverse biological functions, ranging from development to adult physiology, have been attributed to prokineticins. Herein we provide an overview of current knowledge of this interesting pair of regulatory peptides.

Bv8/Prokineticin proteins and their receptors.

The Bv8/Prokineticins (PKs) are a new family of peptides identified in frog, fish, reptiles and mammals that signal through two highly homologous G-protein coupled receptors, PKR1 and PKR2. Bv8/PK proteins possess a unique structural motif comprising five disulfide bonds and a completely conserved N-terminal hexapeptide sequence that is essential for the peptide's biological activities. Over the past few years, several biological functions of Bv8/PK proteins have been elucidated. This review considers all the published data on the action and physiological role of this new biological system implicated in angiogenesis and neurogenesis, in reproduction and cancer and in regulating physiological functions that underly circadian rhythms, such as the sleep/wake cycle, hormone secretion and ingestive behaviors. The high expression level of human Bv8/PK2 in bone marrow, lymphoid organs and leukocytes suggested an involvement of these peptides in hematopoiesis and in inflammatory and immunomodulatory processes. Our review highlights the role of the Bv8/PK and their receptor system in setting the pain threshold under normal and pathological conditions.

The prokineticins: a novel pair of regulatory peptides.

Secreted peptides play broad regulatory roles in brain function and elsewhere in the body. Prokineticins are a pair of newly identified regulatory peptides that signal through two highly homologous G protein-coupled receptors. Prokineticins possess a unique structural motif of five disulfide bonds and a completely conserved N-terminal hexapeptide sequence that is essential to biological activity. Diverse biological functions, including roles in development and cell differentiation, have been assigned to the prokineticins. A network of genes, subject to various transcriptional factors, may functionally converge on the prokineticins as regulatory targets.

Unique expression and regulatory mechanisms of EG-VEGF/prokineticin-1 and its receptors in the corpus luteum.

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) or Prokineticin-1 (PK-1) is a novel cysteine-rich protein that belongs to the AVIT protein family. EG-VEGF/PK-1, described as selective angiogenic mitogen, is widely expressed in different tissues including steroidogenic endocrine glands. This review summarizes the expression and functions of EG-VEGF/PK-1 in corpus luteum (CL)-derived cells: endothelial and steroidogenic cell types. EG-VEGF/PK-1 mRNA is expressed by luteal steroidogenic cells of human, rat and bovine ovaries, but was absent from the luteal Endothelial cells CLEC. Luteal EC expressed high levels of both PK-receptors PK-R1 and PK-R2 - the two G protein-coupled PK-1 receptors. Interestingly, expression of EG-VEGF/PK-1 and VEGF were inversely regulated in human and bovine luteinized granulosa cells. EG-VEGF/PK-1 elevated [3H]-thymidine incorporation, MAPK activation and c-jun/fos mRNA expression and enhanced LEC proliferation. EG-VEGF/PK-1 also inhibited serum starvation-induced apoptosis in these cells. Stress conditions such as serum withdrawal, TNFalpha and chemical hypoxia markedly increase PK-R2 expression, whereas mRNA levels of PK-R1 remain unchanged, implying that the anti-apoptotic effect of PK-1 on LEC may be mediated via PK-R2. Besides its direct mitogenic and anti-apoptotic effects, EG-VEGF/PK-1 elevated VEGF mRNA expression in bovine luteal steroidogenic cells, which possesses only PK-R1. Together, these findings suggest an important role for PK-1 in luteal function by acting as a mitogen and survival factor in LEC. Nevertheless, the inverse regulation of EG-VEGF/PK1 and VEGF mRNA expression by ovarian cells and the distribution of its receptors may suggest that in addition to its angiogenic effects, EG-VEGF/PK-1 may also play other roles in ovary.

An ancient role for a prokineticin domain in invertebrate hematopoiesis.

Hemopoietic development requires firm control of cell proliferation and differentiation. Although recent research has revealed conserved function of transcription factors and signaling pathways regulating lineage commitment in hemopoietic development in Drosophila melanogaster and vertebrates, little is known about hemopoietic cytokines among the invertebrate phyla. In the present study, we show that differentiation and growth of hemopoietic stem cells in vitro from an invertebrate, Pacifastacus leniusculus, require an endogenous cytokine-like factor, astakine, containing a prokineticin (PK) domain. Astakine induces a strong hematopoiesis response in live animals. An astakine homologue was also found in the shrimp, Penaeus monodon. So far, PK domains are only identified in vertebrates, in which they, for example, direct angiogenic growth. Our finding of the first PK-like cytokine characterized from any invertebrate provides novel information concerning the evolution of growth factors and blood cell development.

Molecular cloning of mRNA from toad granular gland secretion and lyophilized skin: identification of Bo8--a novel prokineticin from Bombina orientalis.

Prokineticins are small (approximately 8 kDa), biologically active secretory proteins whose primary structures have been highly conserved throughout the Animal Kingdom. Representatives have been identified in the defensive skin secretions of several amphibians reflecting the immense structural/functional diversity of polypeptides in such. Here we describe the identification of a prokineticin homolog (designated Bo8) from the skin secretion of the Oriental fire-bellied toad (Bombina orientalis). Full primary structural characterization was achieved using a combination of direct Edman microsequencing, mass spectrometry and cloning of encoding skin cDNA. The latter approach employed a recently described technique that we developed for the cloning of secretory peptide cDNAs from lyophilized skin secretion, and this was further extended to employ lyophilized skin as the starting material for cDNA library construction. The Bo8 precursor was found to consist of an open-reading frame of 96 amino acid residues consisting of a putative 19-residue signal peptide followed by a single 77-residue prokineticin (Mr=7990 Da). Amino acid substitutions in skin prokineticins from the skin secretions of bombinid toads are confined to discrete sites affording the necessary information for structure/activity studies and analog design.

EG-VEGF and Bv8. a novel family of tissue-selective mediators of angiogenesis, endothelial phenotype, and function.

Angiogenic molecules are the focus of therapeutic efforts to promote new vessel development in ischemic or damaged tissue and, conversely, to inhibit endothelial cell growth and survival in proliferative disease. Two novel angiogenic mitogens have been characterized recently. Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) and the mammalian homologue of Bombina variegata peptide 8 (Bv8) are endothelial cell mitogens and chemotactic factors with restricted expression profiles and selective endothelial cell activity. These highly related peptides share two cognate G-protein-coupled receptors that are homologous to the neuropeptide Y receptor. The identification of tissue-selective angiogenic factors raises the possibility that other secreted molecules in this class exist. The potential advantage of tissue-specific angiogenic therapeutics may be the reduction of systemic side effects. Additionally, these peptides or their receptors may be attractive targets for inhibition in several disorders.

The AVIT protein family. Secreted cysteine-rich vertebrate proteins with diverse functions.

Homologues of a protein originally isolated from snake venom and frog skin secretions are present in many vertebrate species. They contain 80-90 amino acids, 10 of which are cysteines with identical spacing. Various names have been given to these proteins, such as mamba intestinal protein 1 (MIT1), Bv8 (Bombina variegata molecular mass approximately 8 kDa), prokineticins and endocrine-gland vascular endothelial growth factor (EG-VEGF). Their amino-terminal sequences are identical, and so we propose that the sequence of their first four residues, AVIT, is used as a name for this family. From a comparison of the sequences, two types of AVIT proteins can be discerned. These proteins seem to be distributed widely in mammalian tissues and are known to bind to G-protein-coupled receptors. Members of this family have been shown to stimulate contraction of the guinea pig ileum, to cause hyperalgesia after injection into rats and to be active as specific growth factors. Moreover, the messenger RNA level of one of these AVIT proteins changes rhythmically in the region of the brain known as the suprachiasmatic nucleus. This shows that members of this new family of small proteins are involved in diverse biological processes.