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Molecular

Cloning and Characterization of the Platelet-Activating Receptor Gene Expressed in the Human Heart

617-624

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Tom Sugimoto 1, Hidetsugu Tsuchimochi 1, Christopher G.A. McGregor 3, Hiroyuki Mutoh4, Takao Shimizu 4, and Yoshihisa Kurachi 1.2)

1 Division of Cardiovascular Diseases, Department of Internal Medicine, 2 Department of Pharmacology, and 3 Division of Thoracic and Cardiovascular Surgery, Department of Surgery, Mayo Clinic, Mayo Foundation, Rochester MN 55905 4 Department of Biochemistry, Faculty of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan

Received

October

24,

1992

PAF decreases cardiac contractility and blood pressure. To characterize the cardiac PAF receptor, we screened a human ventricular cDNA library in a low stringency condition, using a PCR product derived from guinea pig lung PAF receptor as a probe. Four clones were obtained and named HVl-4. In Xenopus oocytes injected with cRNA derived from HV3 or 4 but not from HVl or 2, PAF elicited a Ca2+-activated Cl- current. HV3 and HV4 were duplicate clones, encoding a 342 amino-acid polypeptide which was identical to that of the human leukocyte PAF receptor. However, a portion of the 5’ untranslated region of HV3 (or 4) was different from that of the leukocyte receptor cDNA. Northern blotting of human ventricles and atria using the HV3 insert showed a single band of -4 kb. These results suggest a tissue-specific translational mechanism responsible for regulation of the expression of the PAF receptor mRNA in these tissues. 0 1992 Academic

Press,

Inc.

PAF, a phospholipid released from stimulated basophils, platelets, macrophages, and polymorphonuclear neutrophils, produces, even at nanomolar concentrations, dramatic cardiovascular effects such as peripheral vasodilation with hypotension, decrease in cardiac output, increase in vascular permeability, coronary vasoconstriction and arrhythmias ( 1,2). Besides being *To whom correspondence should be addressed. Abbreviations:

PAF, platelet-activating factor; PCR, polymerase chain reaction; cRNA,

complementary RNA. 0006-291X/92

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implicated as a mediator in platelet aggregation, thrombosis, inflammation, asthma, and shock, PAF also plays a major role in the development of cardiac anaphylaxis (3). However, the mechanism through which PAF acts upon the heart is unresolved. Since PAF is a strong aggmgator and activator of platelets and neutrophils, vasoactive substances including thromboxane AZ, leukotriens and histamine which are released from these cells, might mediate some of the effects of PAF on the cardiovascular system (4). However, in isolated perfused hearts, a preparation free of blood cells, PAF still reduces coronary blood flow, has a negative inotropic effect, and produces rhythm disturbances (5). Moreover, in single cardiac cells, PAF modulates K+ channels (6,7). This indicates that PAF could act directly on cardiac myocytes. However, the evidence that PAF receptors exist in the heart has only been indirect, based on the findings that PAF-antagonists can prevent some of the cardiac effects of PAF (8). Pharmacological studies have suggested the existence of two PAF receptor subtypes in cardiac muscle (9). In non-cardiac tissue, specific PAF-receptor sites have been identified by radioligand binding techniques in platelets, leukocytes, lung, and tracheal epithelial cells (2). Furthermore, the cloning and expression of the PAF receptor from the lung and leukocytes (10,ll) unequivocally demonstrated the existence of PAF receptors in these tissues. The present study was undertaken to uncover whether PAF receptors are expressed in the human heart, with special reference to the existence of receptor subtypes. Exuerimental

Procedura

Construc&on of a nrobe bv PCR and screenine of human ventricular cDNA library, Among several oligonucleotides synthesized with reference to the nucleotide sequence of the guinea pig lung PAF receptor cDNA (lo), two oligonucleotides were used to amplify cDNA derived from human ventricle. The sense primer, s’TAGAATTCCAGCCAGAGCCATGGAs’, corresponds to the sequence from -15 nucleotide (nt) to 5 nt, and the antisense primer, s’AACTGCAGGGTGACCTGATGV’, from 882 nt to 844 nt of the guinea pig PAF receptor cDNA (10). PstI site or EcoRI site was added artificially for subsequent cloning. PCR was performed using Taq polymerase with Taq Ampli kit (Perkin Elmer Cents, Norwalk, CT). PCR consisted of 30 cycles of 1 min denaturing at 94’C, 2 min annealing at 55’C, and 3 min extension at 72’C. The PCR products were then analyzed by agarose gel and the band of expected size (897 b.p.) was purified and digested with PstI and EcoRI, and then subcloned into pBluescript II SKplasmid (Stratagene, LaJolla, CA), which was subjected to sequence analysis. After partial sequencing, the PCR product was used as a probe for screening a human ventricle cDNA libtary (Stratagene). 2 x 106 Phage clones were screened with a s2P-labelled PCR product. Hybridization was conducted in 5 x SSC (the composition of SSC is 0.15 M NaClO.015 M sodium citrate, pH 7.0), 50% formamide, 0.08% bovine serum albumin, 0.08% Ficoll, 0.08% polyvinylpyrrolidone, 0.1% sodium dodecyl sulfate (SDS), 0.25 M NaH2PO4,250 pg/ml denatured salmon sperm DNA, at 37°C for 17 hours. Filters were washed twice with 2 x SSC, 0.1% SDS at room temperature for 20 min, and then exposed to x-ray film overnight at -8O’C with intensifying screen. These procedures were repeated until positive clones were obtained. other conventional DNA recombinant techniques were as described (12). Functional exoression of human e tricular PAF receptor in Xenonus oocvtes, Each phage clone was co~vted to plasmid by a rescue excision protocol. Each plasmid obtained was transcribed in vitro in the presence of cap analogue, 7 methyl GpppG (New England Biolab, Bevrly, MA) by Ts or T7 RNA polymerase after digestion with XhoI and NotI, 618

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respectively. These transcripts were dissolved in sterile water, and injected to manually defolliculated oocytes (about 2.5 ng - 5 ng in 25 nl/oocyte). Injected oocytes were incubated in a modified Barth solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHC@, 0.82 mM MgS04,0.33 mM Ca(NC&, 0.41 mM CaQ7.5 mM Tris, pH 7.6) at 19°C for 2-3 days. Electrophysiological studies using the voltage clamp technique were undertaken to determine which transcript could express a functional PAF receptor. The oocytes were maintained at -70 mV in a frog Ringer solution (115 m NaCl, 2 mM KCl, 1.8 mM CaC12,5 mM Hepes, pH 7.4) containing 0.1% fatty acid free bovine serum albumin (Sigma, Fraction No.5 Lot. 119F9306.. St. Louis, MO), and were monitored for an electrophysiological response caused by activation of the Ca2+-activated chloride channel (L-& elicited by PAF applied to the bath solution. Northern blot analvs’ Pieces of can& tissue (right and left ventricle, right atrium) were removed (with family consensus), from a 60-year old healthy male who died in a traffic accident and was a donor for kidney transplantation. Macroscopic examination of the heart revealed no gross abnormality. RNAs were extracted by the guanidium thiocyanate method (13). Aliquots of 10 pg of RNA were separated by electrophoresis in 1.O % agarose gel and blotted onto a Hybond-N nylon membrane (Amersham, Arlington Heights, IL). The agarose gel contained 0.04 M 3-[N-Morpholinolpropane sulfonic acid (MOPS, Sigma), 10 mM sodium acetate, 2.2 M formaldehyde, and 1 mM EDTA. Hybridization was performed at 42°C for 17 hours in the same solution as used in the cDNA library screening. Final washing of the filter was performed at 60°C for 15 min in 0.05 x SSC containing 0.1% SDS. The filter was exposed to x-ray film at -8O’C for four days with an intensifying screen. DNA seauencine. The clones of PAF, which were functional when injected into Xenopus oocytes, were digested with several restriction enzymes, and the fragments were subcloned into pBluescript II SK- or Ml3 mp18 or 19 to obtain continuous overlapping subclones. Sequencing was performed in both orientations using a sequencing kit (USB, Cleveland, OH). Other clones were also sequenced completely or partially using synthetic oligoprimers. Miscellaneous. PAF (C- 16) and Lyso PAF were purchased from Cayman Chemical Company (Ann Arbor, MI). WEB 2086 was a gift from Boehringer Mannheim (Mannheim, Germany).

To construct a probe with which to screen the cDNA library, we used two oligonucleotides (see Methods) corresponding to the ATG initiation site and the putative VIth transmembrane region of the guinea pig lung PAF receptor cDNA. Using these probes, we amplified the human ventricular cDNA library and obtained a PCR product. Analysis of the PCR product by agarose gel revealed a single clear band with an expected size of 897 b.p.. The fragment was subcloned and subjected to partial sequencing, which revealed a high degree of identity (21 out of 22 amino acids of transmembrane domain I) with the guinea pig lung PAF receptor cDNA. In order to obtain a clone which encodes a complete amino acid sequence of the ventricular PAF receptor, we screened a human ventricular cDNA library using the PCR product as a probe. A low stringency condition (the temperature of hybridization was 37’C, and the filters were washed at room temperature ) was employed to obtain possible PAF receptor subtypes. After screening of 2 x 106 human ventricular cDNA clones, four clones were obtained. These phage clones were converted to plasmids by rescue excision to generate HV 1,2,3 and 4. The analysis of the insert size and restriction map suggested that I-IV2 and HV4 were duplicates of 619

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HVl and HV3, respectively. HV3 and I-IV4 had the longest insert (1.3 kb), whereas HVl and HV2 had a shorter insert (0.4 kb). To confirm that these clones were actually encoding functional PAF receptors, bidirectional complementary RNAs (cRNA) were made from each clone and injected into Xenopus oocytes. Oocytes injected with cRNA made by T7 RNA polymerase from Not I cut HV3 or HV4, but not HVl and 2, elicited a Ca2+-dependent Cl- current (Ica.ct) in response to PAP added to the bathing solution. When oocytes injected with 2.5 ng of I-IV3 transcript were voltage-clamped at -70 mV, lo-8 M PAF evoked an inward Ica.ct with a peak amplitude of 0.5 - 1.0 pA (Fig. 1 Aa). The current response was inhibited by -80% with the PAF antagonist, WEB 2086 (10-s M) (Fig. 1 Ab). Lyso PAF (10-s -10-e M), an inactive analogue of PAF, did not elicit the current response (Fig. 1 Aa). Fig. 1B shows the peak amplitude of PAF (10 nM)-induced ka.cl at -70 mV in the oocytes injected with various amounts (0.25-2500 pg/cocyte) of cRNA of HV3. The oocytes injected with 25 ng of HV3 cRNA clearly induced Ica.a in response to 10 nM PAF. The peak amplitude of the PAF-induced Ica.cl increased as the amount of cRNA injected into oocytes was increased. Fig. 1C

A

a

LysoPAF 1 .E!Y PAF 10 nM 7

b

1 100 nA 1 min

PAF 10 nM WEB2086 10 nM

_1 100 nA 1 min

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PAF (M) cRNA (pg/oocyte) Figure 1. El ectro~ hvs il_’ o omxl analvsisof the HV3 clone exuressedin Xenouus oocv&s. (A) The in vitro svnthesizedcRNA 8.5 ne) from HV3 was iniected intc occvtes. After incubation at 19’C for two days, oocyteswere voltage clamped at -70 mV. The inwarda- currentselicited by PAF addedto the bathing solution were monitored. The bar above the current traceindicatesthe drug application. a) Electrophysiologcalresponsesto the application of 10-sM PAF. b) Electrophysiologicalresponseto 10-sM PAF after preincubationwith 10-aM WEB 2086. (B) Electmphysiologicalresponsesasa function of the amount of HV3 cRNA injected into oocytes. Vertical bars denote SE. (n = 5-7). (C) Concentration-responsecurve of PAF in eliciting electrophysiologicalresponsein oocytes. Vertical bars denote S.E. (n = 5-8). 620

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of PAF (lo-13 - 10-G M) in eliciting

Ica,cl at -70 mV

in oocytes injected with 2.5 ng of HV3 cFWA. The threshold concentration of PAF for inducing 1~~~1was -10-11 M. The half maximal response (EDso) occurred at -10-s M PAF. Similar results were obtained in oocytes injected with HV4. These values are in agreement with those reported for the guinea-pig lung and human leukocyte PAF receptors (14,15). These results indicate that HV3 and 4 were cDNA which encoded functional PAF receptors. Fig. 2 shows the nucleotide sequence of 1530 bases for HV3 cDNA. The 3’ non-coding sequence lacks a poly (A) tail and the consensus polyadenylation signal, indicating that HV3 is a partial cDNA. Hydrophobicity analysis of the deduced amino acid sequence revealed seven hydrophobic stretches that could represent transmembrane domains, a common feature to the superfamily of G protein-coupled receptors. A single amino acid sequence was determined which was identical to that of the human leukocyte PAF receptor (11). However, HV3 possessed a second ATG at - 128 nt upstream of the initial methinine codon for the PAF receptor. The amino

Figure 2. m ammo me HVS. The nucleotide sequence is numbered starting with the first methionine of the longest reading frame. The deduced amino acid sequence is shown beneath the nucleotide sequence. Putative transmembrane domains are indicated by solid bars upper the amino acid sequence. The 5’ non-coding region which was different from that of human leukocyte PAF receptor is indicated by a single (or dotted) underline. Double underline indicates a second open reading frame. 621

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acid sequence following this upstream ATG consisted of an open reading frame of 30 codons. This second open reading frame is unusually long when compared to other rhodopsin type receptors such as rat D2 receptor ( 16), hamster l32 receptor (17), as well as guinea pig lung and human leukocytes PAF receptors (10,ll). Sequencing of other clones revealed that HV4 was identical to HV3, and that HVl was to HV2. HVl (or 2) encoded the coding region from the transmembrane III to IV of HV3 (or 4). These results may indicate that these clones were duplicates arising from the amplification of the cDNA library. Thus, we could not identify any other structurally-different PAF receptor from that expressed in the human leukocyte. However, we found that HV3 has a 5’ non-coding region different from that of human leukocyte PAF receptor cDNA. HV3 and leukocyte PAP receptor cDNAs encode a common 5’ non-coding sequence until the position of -41 nt. However, a different 5’ non-coding sequence from that of leukocyte PAP receptor cDNA was present from the position of -42 nt. This substitution of a portion of the 5’ non-coding region may have several physiological consequences: [l] The Southern blot analysis using the human DNA showed that there is a single copy of the PAF receptor gene (II. Mutoh and T. Shimizu, unpublished data), which may indicate that this substitution of the 5’ noncoding region is possibly generated by an alternative splicing event. Thus, it is suggested that different transcriptional mechanisms produce PAF receptor mRNAs in cardiac tissue and leukocytes. [2] The substitution generates an unusually long (30 codons) second open reading frame, which may affect the efficiency of translation as reported for the human & adrenergic receptor (18). It was shown that the human & adrenergic receptor has a second upstream ATG and a relatively long (19-codon) open reading frame in the 5’ non-coding region. When cRNA of the mutated l32adrenergic receptor which lacked this upstream second open reading frame was translated in an in vitro rabbit reticulocyte system or in oocytes, the production of the receptor protein was approximately lo-fold higher than that of the wild type. This observation may indicate the importance of the presence of a second reading frame in the 5’ noncoding region in the regulation of its translation in vivo. Therefore, the second ATG and a 30-codon open reading frame in HV3 may possibly play an important role in regulating the efficiency of translation of the PAF receptor mRNA in the heart. Further studies using mutants in the 5’ non-coding region of I-IV3 are needed to delineate the importance of this region in the control of translation. To determine whether HV3 is actually expressed in the heart, we performed Northern blot analysis using RNAs extracted from human atrium and ventricle. As shown in Fig. 3, one clear band of -4.0 kb was demonstrated in the left and right ventricle as well as the atrium, indicating that I-IV3 was indeed expressed in human cardiac tissue, and there were no multiple transcripts as reported previously (10). Since we did not examine the cellular localization of HV3 messengers using the in situ hybridization technique, we could not specificaly determine which cell type in the cardiac tissue, i.e. cardiac myocytes, coronary arterial smooth muscle cells, or endothelial cells, actually expresses HV3. 622

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Figure 3. Northern blot &sis of HV3 Eachlane contained 10 pg of RNA from human right atrium (lane l), right ventricle (lane 2) and left ventricle (lane 3). The positions of 28s and 18s rRNA are indicated.

Although we screened the human ventricular cDNA library under a low stringency condition, only a single type of PAF receptor cDNA was obtained. Since the amino acid sequences of HV3 and the human leukocyte PAF receptor were exactly identical, it is unlikely that the different PAF receptor subtypes might be expressed in a tissue specific manner. In conclusion, our study showed that the PAF receptor protein expressed in human cardiac tissue is exactly the same as that of human leukocytes. The 5’ non-coding region of cardiac PAF receptor cDNA is different from that found in leukocytes suggesting the presence of tissue specific regulatory mechanisms in the expression of this protein. Acknowledgments The authors thank Dr. Andre Terzic (Mayo Foundation) for his critical reading of this manuscript and Ms. Elizabeth Erlandson for her technical assistance. This work was supported by NIH ROl HL47360-01 to Y.K. and was done during the tenure of an Established Investigatorship of the American Heart Association to Y. K.

1. 2. 3. 4. 5. 6. ii: 9. 10.

Vargaftig, B.B., Chignard, M., Benveniste, J., Lefort J. and Wal, F. (1981) Ann. NY Acad. Sci. 370: 119-137. Snyder, F. (1990) Am. J. Physiol. 259: C697-C708. Braquet, P., Touqui, L., Shen, T.Y. and Vargaftig, B.B. (1987) Pharmacol. Rev. 39: 97-145. Levi, R., Burke, J.A., Guo, Z-G., Hattori, Y., Hoppens, C.M., McManus, LM, Hanahan, DJ and Pinckard, RN (1984) Circ. Res. 54: 117-124. Flores, N.A. and Sheridan, D.J. (1990) Br. J. Pharmacol. 101: 734-738. Wahler, G.M., Coyle, D.E. and Sperelakis, N. (1990) Mol. Cell. B&hem. 93: 69-76. Nakajima, T., Sugimoto, T. and Kurachi, Y. (1991) FEBS L&t. 289: 239-243. Sir&, A-L. and Feuerstein, G. (1989) Am. J. Physiol. 257: H25-H32. Hu, W. and Man, R.Y.K. (1991) Br. J. Pharmacol. 104: 773-775. Honda, Z., Nakamura, M., Miki, I., Minami, M., Watanabe, T., Seyama, Y., Okado, H., Toh, H., Ito, K., Miyamoto, T. and Shimizu, T. (1991) Nature 349: 342-346. 623

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11. 12. 13. 14. 15. 16. 17. 18.

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Nakamura, M., Honda, Z., Izumi, T., Sakanaka, C., Mutoh, H., Minami, M., Bito, H., Seyama, Y., Matsumoto, T., Noma, M. and Shimizu, T. (1992) J. Biol. Chem. 266: 20400-20405. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ED., (Cold Spring Harbor Laboratory Press, New York). Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) Biochemistry 18: 5294-5299. Travers, J.B., Li, Q., Kniss, D.A. andFerte1, R.H. (1989) J. Immunol. 143: 3708-3713. Kroegel, C.; Pleass, R., Yukawa, T., Chung, K.F., Westwick, J. and Barnes, P.J. (1989) FEBS Lett. 243: 41-46. Bunzow, J.R., Van Tol, H.H.M., Grandy, D.K., Albert, P., Salon, J., Christie, M., Machida,‘C.A., Neve, K.A. and Civelli, 0. (1988) Nature 336: 783-787. Dixon, R.A., Kobilka, B.K., Strader, D.J., Benovic, J.L., Dohlman, H.G., Frieile, T., Bolanowski, M.A., Bennett, C.D., Rands, E., Diehl, R.E., Mumford, R.A., Slater, E.E., Sigal, I.S., Caron, M.G., Lefkowitz, R.J. and Strader, C.D. (1986) Nature 321:75-79. Kobilka, B.K., MacGregor, C., Daniel, K., Kobilka, T.S., Caron, M.G., Lefkowitz, R.J. (1987) J. Biol. Chem., 262: 15796-15802.

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Molecular cloning and characterization of the platelet-activating factor receptor gene expressed in the human heart.

PAF decreases cardiac contractility and blood pressure. To characterize the cardiac PAF receptor, we screened a human ventricular cDNA library in a lo...
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