Identification and Molecular Characterization of Two Closely Related G Protein-coupled Receptors Activated by Prokineticins/Endocrine Gland Vascular Endothelial Growth Factor

Abstract

We previously described two mammalian secreted proteins, prokineticin 1 and prokineticin 2, that potently contract gastrointestinal smooth muscle. Prokineticin 1 has also been shown to promote angiogenesis by stimulating proliferation, migration, and fenestration of endocrine organ-derived endothelial cells. Here we report the cloning and characterization of two closely related G protein-coupled receptors as receptors for prokineticins. Expression of prokineticin receptors in heterologous systems shows that these receptors bind to and are activated by nanomolar concentrations of recombinant prokineticins. Activation of prokineticin receptors leads to mobilization of calcium, stimulation of phosphoinositide turnover, and activation of p44/p42 MAPK signaling pathways that are consistent with the effects of prokineticins on smooth muscle contraction and angiogenesis. mRNA expression analysis reveals that prokineticin receptors are expressed in gastrointestinal organs, endocrine glands, and other tissues. Diseases involving altered gastrointestinal (GI)1 motility are among the most common human disorders (1, 2). Understanding the physiological functions and mechanisms of action of GI motility regulatory factors is a prerequisite for developing effective treatments for GI motility disorders (3, 4). We have previously discovered two mammalian proteins, prokineticin 1 and 2 (PK1 and PK2, respectively), that are potentially important regulators of GI motility (5). Prokineticins are ϳ10-kDa cysteine-rich secreted proteins expressed in the GI tract and several other tissues. Recently a second function for PK1 was reported. This protein, which LeCouter et al. (6) named endocrine gland vascular endothelial growth factor, stimulates cell proliferation, migration, and fenestrations in several endothelial cell lines derived from endocrine glands. In addition, expression of PK1 from recombinant adenovirus injected into the mouse ovary stimulates angiogenesis (6). Thus, prokineticins regulate diverse biological functions that include contraction of GI smooth muscle and angiogenesis. Based on pharmacological evidence, we have demonstrated that a receptor(s) for prokineticins belongs to the family of G protein-coupled receptors (GPCRs) (5). Here we report the cloning of two closely related G protein-coupled receptors as receptors for prokineticins. EXPERIMENTAL PROCEDURES Cloning of Prokineticin Receptor 1 (PKR1) and ProkineticinReceptor 2 (PKR2) cDNAs-Human PKR1 and PKR2 sequences were identified in human sequence genome searches as described previously (7). Fulllength cDNAs were cloned from Marathon RACE Ready cDNA (CLON-TECH) by PCR. The PKR1 gene was amplified from testis Marathon RACE Ready cDNA using PCR and the following oligonucleotide primers: 5Ј-ggtgacatcagccttgcagacattgccc and 5Ј-ATGTGCATCCAAGCACA-CTAGTCAGTGTCC. This was followed by nested PCR using the following oligonucleotide primers: 5Ј-CACCATGGAGACCACCATGGG-GTTCATG and 5Ј-ATGTGCATCCAAGCACACTAGTCAGTGTCC. PKR2 was amplified from pooled testis and fetal brain Marathon...

Bv8, the amphibian homologue of the mammalian prokineticins, modulates ingestive behaviour in rats

“…Receptors for PK-1 and PK-2 have been identified in humans, mice (Lin et al, 2002) and rats (Masuda et al, 2002). These receptors, named prokineticin receptor 1 and 2 (PKR-1 and PKR-2), belong to the family of G-protein-coupled receptors, share approximately 85% amino-acid identity and are about 80% identical to the previously described mouse orphan receptor gpr73 (Parker et al, 2000).…”

Prokineticins are neuroprotective in models of cerebral ischemia and ischemic tolerance in vitro

“…The mammalian prokineticin family includes two small bioactive peptides (8e12 kDa) that stimulate guinea-pig ileum contraction termed prokineticin 1 (PK1) (also known as endocrine gland-vascular endothelial growth factor, EG-VEGF) and PK2 (the mammalian analog of Bv8). These two peptides are highly conserved across species (LeCouter et al, 2004;Giannini et al, 2009) and activate two highly homologous receptors, prokineticin receptor 1 (PKR1) and PKR2 (Negri and Lattanzi, 2011), which are coupled to a variety of G proteins and hence to intracellular Ca 2þ mobilization and activation of the ERK1/2 and Akt pathways (Lin et al, 2002;Masuda et al, 2002;Soga et al, 2002). Prokineticins (especially PK2) and their receptors are largely expressed in CNS (Cheng et al, 2006) and regulate multiple biological functions in the CNS including hyperalgesia (Mollay et al, 1999), neurogenesis in the olfactory bulb (Ng et al, 2005), neuronal survival (Melchiorri et al, 2001) and inflammation (Martucci et al, 2006).…”

Prokineticin 2/Bv8 is expressed in Kupffer cells in liver and is down regulated in human hepatocellular carcinoma

“…Prokineticins are a family of peptides characterized by a common AVIT motif, and 10 conserved cysteine residues [24][25][26] . Prokineticins are not structurally related to VEGF and mediate their biological activity by selectively acting on two G protein coupled receptors, prokineticin receptor 1 and 2 (PKR1 and PKR2), for which they have similar affinities [24,26] . Activation of PKRs triggers multiple signaling pathways, such as calcium mobilization, phosphoinositol turnover, and activation of Akt kinase and mitogen-activated protein kinase (MAPK) [27] .…”

“…In addition, in a mouse model of myocardial infarctions, PK2/Bv8 protected cardiomyocytes by increasing neovascularization [23] . Prokineticins are a family of peptides characterized by a common AVIT motif, and 10 conserved cysteine residues [24][25][26] . Prokineticins are not structurally related to VEGF and mediate their biological activity by selectively acting on two G protein coupled receptors, prokineticin receptor 1 and 2 (PKR1 and PKR2), for which they have similar affinities [24,26] .…”