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J. Biol. Chem., Vol. 277, Issue 22, 19276-19280, May 31, 2002

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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^**, and Qun-Yong Zhou^¶

From  Tularik Inc., South San Francisco, California 94080 and the ^¶ Department of Pharmacology, University of California, Irvine, California 92697

Received for publication, March 4, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 ~10-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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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). Full-length cDNAs were cloned from Marathon RACE Ready cDNA (CLONTECH) by PCR. The PKR1 gene was amplified from testis Marathon RACE Ready cDNA using PCR and the following oligonucleotide primers: 5'-ggtgacatcagccttgcagacattgccc and 5'-ATGTGCATCCAAGCACACTAGTCAGTGTCC. This was followed by nested PCR using the following oligonucleotide primers: 5'-CACCATGGAGACCACCATGGGGTTCATG and 5'-ATGTGCATCCAAGCACACTAGTCAGTGTCC. PKR2 was amplified from pooled testis and fetal brain Marathon RACE Ready cDNA using PCR and the following oligonucleotide primers: 5'-CACCATGGCAGCCCAGAATGGAAAC and 5'-TGGGTCACTTCAGCCTGATACAGTC. These PCR products were cloned into pcDNA3.1D/V5-His-TOPO (Invitrogen) and confirmed by DNA sequencing.

Analysis of Prokineticin Receptor Signaling-- An aequorin-based luminescent assay for calcium mobilization was used essentially as described previously to measure mobilization of intracellular Ca²⁺ (7). Chinese hamster ovary (CHO) cells were transiently co-transfected with pcDNA3-Aequorin and either PKR1 or PKR2 expression plasmids using LipofectAMINE (Invitrogen). Measurements were recorded using a TD20/20 luminometer (Turner Designs).

Prokineticin-stimulated inositol phosphate production was measured in COS-7 cells following transient transfection of PKR1 and PKR2. Quantification of inositol phosphate was performed as described previously (8).

Activation of p44/p42 mitogen-activated protein kinase (MAPK) was monitored by Western blotting with anti-phospho-MAPK antibody (Cell Signaling Technology). HEK293 parental cells and HEK293 cells stably expressing PKR1 or PKR2 were plated in 24-well tissue culture dishes and serum-starved (Dulbecco's modified Eagle's medium containing 0.1% fetal bovine serum) for 24 h. Next the spent medium was aspirated, recombinant prokineticins were added, and total cellular extracts were prepared in Cell Lysis Buffer (Cell Signaling Technology). Twenty micrograms of total protein were loaded per lane and were separated on a denaturing polyacrylamide gel.

Iodination of Recombinant Prokineticin 1 and Receptor Radioligand Binding-- Recombinant human PK1 and PK2 were purified from Escherichia coli and refolded as described previously (5). Iodination of PK1 was performed by incubating 4.0 µg of PK1 with 50 µg of Iodogen in 50 µl of 0.5 M phosphate-buffered saline, pH 7.2, for 15 min at room temperature. The reaction was stopped by transferring the mixture to a microcentrifuge tube containing 100 µl of a phosphate-buffered saline solution containing 1 mM NaI and 0.1% bovine serum albumin. Free iodine was removed by gel filtration on Bio-Gel P2, and the radioactivity in the void volume was measured. Assuming that all radioactivity had been incorporated into the 3.0 µg of PK1 (75% recovery), we calculated the specific radioactivity to be 520-1043 Ci/mmol.

For receptor radioligand binding, COS-7 cells in 100-mm dishes were transiently transfected with PKR1 or PKR2 expression plasmid with the LipofectAMINE method (9). Transfected cells were split into 24-well dishes, and binding was carried out 48-72 h after transfection. For saturation binding, 0.03-8.0 nM ¹²⁵I-labeled PK1 was used. Incubation of radioligand was in -minimum Eagle's medium containing 0.1% bovine serum albumin for 4 h at 4 °C. Cells were extensively washed and lysed with 1 N NaOH. Radioactivity was measured in a  counter and analyzed with Prism software. For displacement experiments, various dilutions of unlabeled recombinant prokineticins were added to compete with ¹²⁵I-labeled PK1 (2.0 nM) for binding to cells. Nonspecific binding was defined in the presence of 500 nM unlabeled PK1.

Analysis of PKR1 and PKR2 mRNAs in Tissues-- Multiple tissue cDNA panels (CLONTECH) and FirstChoice PCR-Ready cDNA (Ambion) were used as templates in PCRs with primers that amplify only PKR1 or PKR2. One nanogram of cDNA was used per reaction. PKR1 was amplified using the oligonucleotide primers 5'-ggtgacatcagccttgcagacattgccc and 5'-ATGTGCATCCAAGCACACTAGTCAGTGTCC for 40 cycles. The reactions were diluted 50-fold in water, and 5 µl were used in a nested PCR amplification (50-µl total reaction volume) using the oligonucleotide primers 5'-ATTCTGAGCAGTGTTGCTCACAGCACCAC and 5'-ATGTGCATCCAAGCACACTAGTCAGTGTCC for an additional 14 cycles. PKR2 was amplified using the oligonucleotide primers 5'-CACCATGGCAGCCCAGAATGGAAAC and 5'-TGGGTCACTTCAGCCTGATACAGTC for 40 cycles. The reactions were diluted 50-fold in water, and 5 µl were used in a nested PCR amplification (50-µl total reaction volume) using the oligonucleotide primers 5'-CAGTTTCACACCCAACTTTAATCCACCCCAAGACCATG and 5'-ATGTGCATCCAAGCACACTAGTCAGTGTCC for an additional 14 cycles.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of Two G Protein-coupled Receptors as Receptors for Prokineticins-- We previously described pharmacological evidence that the receptor(s) for PK1 and PK2 belongs to the GPCR superfamily (5). To identify putative prokineticin receptors, we cloned several novel members of the GPCR superfamily identified by a hidden Markov model (10). The function of a subset of these GPCRs was tested following transient transfection of their corresponding cDNAs into CHO cells. Intracellular Ca²⁺ mobilization was measured using recombinant PK1 (50 nM) as a ligand. Of the GPCRs tested, only two produced PK1-mediated calcium mobilization under these conditions (data not shown). These human receptors are ~80% identical to the previously described mouse orphan receptor GPR73 (11) and were tentatively designated GPR73a and GPR73b. GPR73a and GPR73b share ~85% amino acid identity and are most divergent in their N-terminal sequences (Fig. 1), with the N terminus of human GPR73a more closely related to mouse GPR73 than to human GPR73b. GPR73a was localized previously to human chromosome region 2p14 (11), and GPR73b was localized in silico to chromosome region 20p13.

View larger version (72K): [in this window] [in a new window]   Fig. 1.   Sequence alignment of human GPR73a (hGPR73a), human GPR73b (hGPR73b), and mouse GPR73 (mGPR73) (GenBankTM accession number NP_067356) proteins. Dark gray boxes indicate amino acid identity. Putative transmembrane domains are overlined.

Both GPR73a and GPR73b were activated by recombinant PK1 and PK2 at nanomolar concentrations in transiently transfected CHO cells (Fig. 2, A and B). GPR73a was stimulated by PK1 and PK2 with EC₅₀ (concentration for half-maximal response) values of 2.2 ± 0.7 and 4.3 ± 1.3 nM (mean ± S.E., n = 3), respectively. GPR73b was stimulated by PK1 and PK2 with similar EC₅₀ values of 7.0 ± 1.8 and 7.3 ± 2.8 nM (mean ± S.E., n = 3), respectively. Similar EC₅₀ values were obtained with CHO cells stably expressing GPR73a and GPR73b (data not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Signaling of GPR73a and GPR73b. A and B, dose-response curves of Ca2+ mobilization in response to prokineticin 1 and 2 in CHO cells transiently co-transfected with the bioluminescent Ca2+ sensor aequorin and GPR73a (A) and GPR73b (B) expression plasmids. Results from one of three independent experiments are shown. RLU, relative light unit. C, dose-response curve of phosphoinositide turnover in COS-7 cells that transiently express GPR73a and GPR73b. Results from one of three independent experiments are shown. D, time course of activation (phosphorylation) of p44/p42 MAPK following addition of 12.5 nM PK1 (left panels) or 12.5 nM PK2 (right panels) in parental HEK293 (top panels) cells or HEK293 cells stably expressing GPR73a or GPR73b (middle and bottom panels).

Coupling of Prokineticin Receptors to G_q-- Because both G_q- and G_i/o-coupled receptors can mediate intracellular calcium mobilization, we further dissected the signaling pathways activated by GPR73a and GPR73b by analyzing the pertussis toxin sensitivity of the calcium response evoked by these two receptors. PK1- and PK2-induced calcium mobilization was not affected by pertussis toxin treatment in transiently transfected CHO cells (data not shown), suggesting that GPR73a and GPR73b couple exclusively to G_q rather than to G_i/o. Furthermore PK1 produced a dose-dependent increase in total inositol phosphate concentration in COS-7 cells transiently transfected with GPR73a and GPR73b (Fig. 2C). The EC₅₀ values for stimulation of inositol phosphate production were 9.0 ± 1.9 nM (mean ± S.E., n = 3) and 29.7 ± 4.6 nM (mean ± S.E., n = 3) for GPR73a and GPR73b, respectively. The maximal response was about 2-fold higher in COS-7 cells transfected with GPR73a when compared with cells transfected with GPR73b. The difference may result from the apparently higher expression level of GPR73a in transfected cells when the same amount of GPR73a and GPR73b expression vector DNA was transfected (see receptor binding studies). Finally neither GPR73a nor GPR73b had an effect on stimulation or inhibition of adenylate cyclase (data not shown).

Activation of Prokineticin Receptors Induces Phosphorylation of p44/p42 Mitogen-activated Protein Kinase-- Since activation of MAPK is critical for angiogenesis (12), we next tested whether the stimulation of GPR73a and GPR73b by PK1 and PK2 activated the p44/p42 MAPK. HEK293 cells stably expressing either GPR73a or GPR73b were incubated with PK1 or PK2 for various time intervals, and phosphorylation of p44/p42 MAPK was detected using a specific anti-phospho-p44/p42 MAPK antibody. PK1 induced a sustained phosphorylation of p44/p42 MAPK in GPR73a-transfected cells but not in GPR73b-transfected cells (Fig. 2D). PK2 also stimulated phosphorylation of p44/p42 MAPK in both GPR73a- and GPR73b-expressing cells although to a lesser extent than that seen with PK1 and GPR73a-expressing cells (Fig. 2D).

Binding of ¹²⁵I-Labeled PK1 to Cells That Express Prokineticin Receptors-- To determine the binding affinity of GPR73a and GPR73b for PK1 and PK2, we performed receptor binding assays on transiently transfected cells. Mock-transfected COS-7 cells did not exhibit detectable levels of specific binding of ¹²⁵I-labeled recombinant human PK1. Binding of ¹²⁵I-labeled PK1 to COS-7 cells that expressed GPR73a was saturable, specific, and with high affinity (K_d = 12.3 ± 4.2 nM, B_max = 11.2 ± 2.27 fmol/10⁵ cells, n = 3). Expression of GPR73b conferred a slightly higher ¹²⁵I-labeled PK1 binding affinity (K_d = 1.8 ± 0.1 nM, n = 3) to the transfected cells, but the receptor level (B_max = 4.39 ± 1.07 fmol/10⁵ cells, n = 3) was slightly lower than that observed for GPR73a. These data indicated that when the same amount of GPCR expression vector DNA was used in transfections, GPR73a yielded about a 3-fold higher receptor expression level than did GPR73b.

The specific binding of ¹²⁵I-labeled, recombinant PK1 to COS-7 cells expressing GPR73a and GPR73b was inhibited by nanomolar concentrations of unlabeled recombinant PK1 and PK2. For GPR73a, the IC₅₀ (concentration for half-maximal inhibition) values were 44.2 ± 4.1 nM (n = 3) and 13.5 ± 0.8 nM (n = 3) for PK1 and PK2, respectively, while for GPR73b, the IC₅₀ values were 34.2 ± 9.0 nM (n = 3) and 4.55 ± 1.40 nM (n = 3) for PK1 and PK2, respectively (Fig. 3, A and B). These results indicated that both PK1 and PK2 had slightly higher binding affinities for GPR73b compared with GPR73a and that PK2 had a slightly higher affinity for both receptors when compared with PK1. On the basis of these receptor signaling and binding results, GPR73a and GPR73b were renamed PKR1 and PKR2, respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Binding of 125I-labeled recombinant prokineticin 1 to COS-7 cells transiently transfected with GPR73a and GPR73b. A, Saturation binding (0-8 nM 125I-labeled recombinant human prokineticin 1). Competition binding of 2 nM 125I-labeled prokineticin 1 by unlabeled prokineticin 1 and prokineticin 2 to GPR73a (B) and GPR73b (C). Results shown are representatives of three independent experiments in triplicates.

Prokineticin Receptors Are Expressed in Diverse Tissues-- To analyze the tissue distribution of PKR1 and PKR2 mRNAs, we performed reverse transcription-PCR analysis on a panel of tissue cDNAs. Both PKR1 and PKR2 were expressed in the small intestine as well as other tissues (Fig. 4). To define more precisely the distribution of PKR1 and PKR2 expression in the digestive tract, reverse transcription-PCR was carried out on cDNA prepared from specific regions of the digestive tract. PKR1 mRNA was localized to the stomach, throughout the small intestine and colon, and to the rectum (Fig. 4). PKR2 mRNA was detected only in the ileocecum. Outside the GI tract, PKR1 and/or PKR2 receptors were expressed in many endocrine tissues, including the thyroid gland, pituitary gland, salivary gland, adrenal gland, testis, and ovary (Fig. 4), where they may function in angiogenesis (6). PKR1 and PKR2 mRNA were both detected in the brain, and PKR1 was also expressed in the spleen, prostate, pancreas, and peripheral blood leukocytes (Fig. 4).

View larger version (63K): [in this window] [in a new window]   Fig. 4.   Distribution of prokineticin receptor mRNAs in human tissues by reverse transcription-PCR. PCR primers that specifically recognize only PKR1 or PKR2 were designed. Reverse transcription-PCR products from human tissues are shown for PKR1 (top panel) and PKR2 (middle panel). Primers that amplify a fragment of actin were used as a positive control (bottom panel). sk., skeletal; sm., small.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We conclude that we have identified two closely related G protein-coupled receptors as receptors for prokineticins/endocrine gland vascular endothelial growth factor. Prokineticin receptors share 85% sequence identity and common signaling pathways, but we do not yet know whether they have overlapping functions. PKR1 is expressed more abundantly in the GI tract compared with PKR2 and thus may be the major receptor mediating the function of prokineticin in the GI system. The presence of both receptors in several endocrine and non-endocrine tissues and their activation of p44/p42 MAPK may be related to the function of prokineticin in angiogenesis (6), which is essential for a variety of physiological and pathological processes (13, 14). The development and analysis of mice lacking one or both of the prokineticin receptors should help determine the physiological functions of PKR1 and PKR2 in GI motility and angiogenesis. Our findings should also help in the understanding of the regulatory roles of prokineticins in GI motility and angiogenesis by allowing the development of selective agonists and antagonists.

	ACKNOWLEDGEMENTS

We thank M. Cheng for technical help and P. Coward, G. Cutler, and T. Hoey for comments on the manuscript.

	FOOTNOTES

^* This work was supported in part by a grant from the National Institutes of Health (to Q.-Y. Z.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF506287 (human PKR1) and AF506288 (human PKR2).

^§ Both authors contributed equally to this work.

A member of the medical scientist training program of University of California, Irvine.

^** To whom correspondence may be addressed: Tularik Inc., Two Corporate Dr., South San Francisco, CA 94080. Tel.: 650-825-7000; Fax: 650-825-7400.

To whom correspondence may be addressed: Dept. of Pharmacology, University of California, 19182 Jamboree Blvd., Irvine, CA 92697. Tel.: 949-824-2232; Fax: 949-824-4855; E-mail: qzhou@uci.edu.

Published, JBC Papers in Press, March 8, 2002, DOI 10.1074/jbc.M202139200

	ABBREVIATIONS

The abbreviations used are: GI, gastrointestinal; PK, prokineticin; PKR, prokineticin receptor; GPCR, G protein-coupled receptor; CHO, Chinese hamster ovary; MAPK, mitogen-activated protein kinase.

	REFERENCES

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				C. M. Lambert, K. K. Machida, L. Smale, A. A. Nunez, and D. R. Weaver Analysis of the Prokineticin 2 System in a Diurnal Rodent, the Unstriped Nile Grass Rat (Arvicanthis niloticus) J Biol Rhythms, June 1, 2005; 20(3): 206 - 218. [Abstract] [PDF]

				J. Chen, C. Kuei, S. Sutton, S. Wilson, J. Yu, F. Kamme, C. Mazur, T. Lovenberg, and C. Liu Identification and Pharmacological Characterization of Prokineticin 2{beta} as a Selective Ligand for Prokineticin Receptor 1 Mol. Pharmacol., June 1, 2005; 67(6): 2070 - 2076. [Abstract] [Full Text] [PDF]

				I. Soderhall, Y.-A Kim, P. Jiravanichpaisal, S.-Y. Lee, and K. Soderhall An Ancient Role for a Prokineticin Domain in Invertebrate Hematopoiesis J. Immunol., May 15, 2005; 174(10): 6153 - 6160. [Abstract] [Full Text] [PDF]

A. R. Albig and W. P. Schiemann Identification and Characterization of Regulator of G Protein Signaling 4 (RGS4) as a Novel Inhibitor of Tubulogenesis: RGS4 Inhibits Mitogen-activated Protein Kinases and Vascular Endothelial Growth Factor Signaling Mol. Biol. Cell, February 1, 2005; 16(2): 609 - 625. [Abstract] [Full Text] [PDF]