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

Daniel Chi-Hong Lin,Clayton M Bullock,Frederick J Ehlert,Jin-Long Chen,Hui Tian,Qun-Yong Zhou

doi:10.1074/jbc.m202139200

Daniel Chi-Hong Lin, Clayton M Bullock + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m202139200

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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.

Highlights

Diseases involving altered gastrointestinal (GI)1 motility are among the most common human disorders [1, 2]
Activation of prokineticin receptors leads to mobilization of calcium, stimulation of phosphoinositide turnover, and activation of p44/p42 mitogen-activated protein kinase (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
We conclude that we have identified two closely related G protein-coupled receptors as receptors for prokineticins/endocrine gland vascular endothelial growth factor

Summary

EXPERIMENTAL PROCEDURES

Cloning of Prokineticin Receptor 1 (PKR1) and Prokineticin Receptor 2 (PKR2) cDNAs—Human PKR1 and PKR2 sequences were identified in human sequence genome searches as described previously [7]. The PKR1 gene was amplified from testis Marathon RACE Ready cDNA using PCR and the following oligonucleotide primers: 5Ј-ggtgacatcagccttgcagacattgccc and 5Ј-ATGTGCATCCAAGCACACTAGTCAGTGTCC. PKR2 was amplified from pooled testis and fetal brain Marathon RACE Ready cDNA using PCR and the following oligonucleotide primers: 5Ј-CACCATGGCAGCCCAGAATGGAAAC and 5Ј-TGGGTCACTTCAGCCTGATACAGTC. These PCR products were cloned into pcDNA3.1D/ V5-His-TOPO (Invitrogen) and confirmed by DNA sequencing. Prokineticin-stimulated inositol phosphate production was measured in COS-7 cells following transient transfection of PKR1 and PKR2. COS-7 cells in 100-mm dishes were transiently transfected with PKR1 or PKR2 expression plasmid with the LipofectAMINE method [9]. The reactions were diluted 50-fold in water, and 5 ␮l were used in a nested PCR amplification (50-␮l total reaction volume) using the oligonucleotide primers 5Ј-CAGTTTCACACCCAACTTTAATCCACCCCAAGACCATG and 5Ј-ATGTGCATCCAAGCACACTAGTCAGTGTCC for an additional 14 cycles

RESULTS

Molecular Identification of Prokineticin Receptors

DISCUSSION