- Email: [email protected]
The orphan G-protein-coupled receptor-encoding gene V28 is closely related to genes for chemokine receptors and is expressed in lymphoid and neural tissues
Gene, 163 (1995) 295-299 © 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50
295
GENE 09054
The orphan G-protein-coupled receptor-encoding gene V28 is closely related to genes for chemokine receptors and is expressed in lymphoid and neural tissues (Degenerate primer PCR; inflammation; human genomic DNA)
Carol J. Raport a, Vicki L. Schweickart", Roger L. Eddy Jr. b, Thomas B. S h o w s b and Patrick W. Gray a alCOS Corporation, Bothell, WA 98021, USA; and bDepartment of Human Genetics, Roswell Park Cancer Institute, Buffalo, NY14263, USA. Tel. (1-716) 845-3108
Received by A.D. Riggs: 23 January 1995; Revised/Accepted: 29 March/30 March 1995; Received at publishers: 15 May 1995
SUMMARY
A polymerase chain reaction (PCR) strategy with degenerate primers was used to identify novel G-protein-coupled receptor-encoding genes from human genomic DNA. One of the isolated clones, termed V28, showed high sequence similarity to the genes encoding human chemokine receptors for monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1~ (MIP-I~)/RANTES, and to the rat orphan receptor-encoding gene RBS11. When RNA was analyzed by Northern blot, V28 was found to be most highly expressed in neural and lymphoid tissues. Myeloid cell lines, particularly THP.1 cells, showed especially high expression of V28. We have mapped V28 to human chromosome 3p21-3pter, near the MIP-I~/RANTES receptor-encoding gene.
INTRODUCTION
G-protein-coupled receptors (GCRs) are cell-surface receptors which recognize extracellular signals and transduce those signals into an intracellular response (Gilman, Correspondence to: Dr. P.W. Gray, ICOS Corporation, 22021 20th Avenue S.E., Bothell, WA 980:21, USA. Tel. (1-206) 485-1900; Fax (1-206) 485-1961; e-mail: pgray~icos.com
Abbreviations: aa, amino acid(s); ATIIR 1, angiotensin II receptor type 1; BLR1, Burkitt's lymphoma receptor 1; bp, base pair(s); DMSO, dimethylsulfoxide; EBI1, Epstein-Barr-induced receptor 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GCR, G protein-coupled receptor; HGMW, Human Gene Mapping Workshop; IL, interleukin; kb, kilobase(s) or 1000 lap; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; NLR, neurolymphatic receptor; nt, nucleotide(s); PBMC, peripheral blood mononuclear cell; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaC1/0.0t5 M Na3"citrate pH 7.6; TM, transmembrane; UTR, untranslated region(s); V28, gene encoding orphan GCR; X, any aa. SSDI 0378-1119(95)00336-3
1987). The signals that act through GCRs are quite diverse, ranging from photons of light to catecholamines to peptides. The receptors themselves share striking structural similarity, containing seven hydrophobic ~-helical membrane-spanning domains with an extracellular Nterminus. Ligand recognition by the receptor leads to G-protein activation and to intracellular responses which include altered cyclic nucleotide metabolism, phospholipid hydrolysis, intracellular ion fluxes and changes in surface ion-channel activity. G-protein-coupled receptors are key regulators of many immune and homeostatic responses. Chemokines, a family of pro-inflammatory peptides, promote chemotaxis in leukocytes by binding to GCRs (Oppenheim et al., 1991). The cloning of two receptor-encoding genes for interleukin 8 (IL-8) (Holmes et al., 1991; Murphy and Tiffany, 1991) first demonstrated that chemokine receptors are members of the GCR family. Subsequently, degenerate PCR techniques were used to clone two addi-
296 tional genes encoding GCRs which recognize the chemokines MIP-I~/RANTES (Neote et al., 1993) and MCP-1 (Charo et al., 1994). We also have utilized a degenerate PCR strategy to identify additional members of the human GCR family which may be involved in immune function. We describe here one such receptor-encoding gene which we called V28.
EXPERIMENTAL AND DISCUSSION
(a) Cloning of the 1/28 gene Human genomic DNA was screened by PCR using degenerate primers to identify genes related to the IL-8 receptors as described by Schweickart et al. (1994). Seven unique PCR fragments were obtained and used to screen a human genomic DNA library (Stratagene) and a human peripheral blood mononuclear cell (PBMC) cDNA plas-
mid library as described by Schweickart et al. (1994). One gene identified in this manner was termed V28. As shown in Fig. 1, the deduced aa sequence of V28 reveals a 355-aa polypeptide which displays the features of other GCRs, including seven hydrophobic domains which may function as TM regions. The V28 protein sequence also contains approx. 20 aa that are highly conserved among members of the GCR superfamily (Probst et al., 1992), though there is no concensus sequence for N-linked glycosylation in the extracellular regions. The V28 sequence includes an aa motif (DRYLAIV) in the second cytoplasmic loop which is particularly conserved among chemokine receptors. V28 shows highest sequence similarity to the rat orphan GCR-encoding gene RBSI I (Harrison et al., 1994), 79% identical at the nt level and 81% identical at the aa level, and to human chemokine receptors for MIP-I~/RANTES and for MCP-1 (see Table I).
V, ACT~GTCTCTGGTAAAGTCTGAGcAGGACAGGGTGGCTGACTGGCAGATCCAGAGGTTCCCTTGGCAGTCCACGCCAGGCCTTCACCATGGATCAGTTCCCTG~TCAGTGACAGAAAAC M D Q F P E S V T E N
120 II
TTT~GTACGATGATT~GCTGAGGCCTGTTATATTGG~ACATCGTGGTCTTT~GACTGTGTTCCTGTCCATATTCTACTCCGTCATCTTTGCCA~CCT~AAATTTGTTG F E Y D D L A E A C Y I G D I V V F G T V F L S I F Y S V I F A I G L V G N L L
240 51
GTAGTGTTTGCCCTCACC~CAGC~G~GCCC~GAGTGTCACCGACATTTACCTCCTG~CC~GCCTTGTCTGATCTGCTGTTTGTAGCCACTT~CCC~CT~ACTCACTATTTG V V F A L T N S K K P K S V T D I Y L L N L A L S D L L F V A T L P F W T H Y L
360 91
TM3 ATAAATGAAAAGGGCCTCCAC~TGCCATGTGCAAATTCACTACCGCCTTCTTCTTCATCGGCTTTTTT~GCATATTCTTCATCACCGTCATCAGCATTGATA~TACCTGGCCATC I N E K G L H N A M C K F T T A F F F I G F F G S I F F I T V I S I D R Y L A I TM4 GTCCT~CCGCC~CTCCATG~C~CCGGACCGTGCAGCATGGCGTCACCATCAGCCTAGGCGTCTGGGCAGCAGCCATTTTGGT~CAGCACCCCAGTTCATGTTCACAAAGCAGAAA V L A A N S M N N R T V Q H G V T I S L G V W A A A I L V A A P Q F M F T K Q K TM~ GAAAATG~TGCCT~GTGACTACCCCGAGGTCCTCCA~AAATCTGGCCCGTGCTCCGC~TGTGGAAACAAATTTTCTTGGCTTcCTACTCCCCCTGCTCATTATGAGTTATTGCTAC E N E C L G D Y P E V L Q E I W P V L R N V E T N F L G F L L P L L I M S Y C Y
480 131 600 171 720 211
TM~ TTCAG~TCATCCAGACGCTGTTTTCCTGC~G~CCAC~GAAAGCCAAAGCCATTAAACTGATCCTTCTGGTGGTCATCGTGTTTTTCCTCTTCTGGACACCCTAC~CGTTATGATT F R I I Q T L F S C K N H K K A K A I K L I L L V V I V F F L F W T P Y N V M I TM7 TTCCTGGAGACGCTT~GCTCTATGACTTCTTTCCCAGTTGTGACATGA~ATCTGAGGCTGGCCCTCAGTGTGACTGAGAC~TTGCATTTAGCCATTGTTGCCTG~TCCTCTC F L E T L K L Y D F F P S C D M R K D L R L A L S V T E T V A F S H C C L N P L
840 251
ATCTATGCATTT~T~GGAG~GTTCAG~GATACCTTTACCACCTGTATGGGAAATGCCTGGCTGTCCTGTGT~GCGCTCAGTCCACGTTGATTTCTCCTCATCTG~TCACAAAGG I Y A F A G E K F R R Y L Y H L Y G K C L A V L C G R S V H V D F S S S E S Q R
1080 331
A~AG~ATGG~GTGTTCTGAGCAGC~TTTTACTTACCACACGAGTGATGGAGATGCATTGCTCCTTCTCTG~GGG~TCCCAAAGCCTTGTGTCTACAGAG~CCT~AGTTCCTG S R H G S V L S S N F T Y H T S D G D A L L L L
1200 355
~CCT~TGCTGACTAGTGAGGAAAGATTTTTGTTGTTATTTCTTACAGGCACAAAATGATGGACCC~TGCACACAAAAC~CCCTAGAGTGTTGTTGAG~TTGTGCTCAAAA~TGA AG~TG~CAAATTG~C~TTTG~TGACAAAGAGTAGACATTTCTCTTACTGCAAATGTCATCAG~CTTTTTGGTTTGCAGATGACAAAAATTC~CTCAGACTAGTTTAGTTAAAT GAG~GTG~TATTGTTCATATTGTGGCAC~GCAAAA~GTGTCTGAGCCCTCAAAGTGAGGGGAAACCA~GCC~AGCC~GCTAG~TTCCCTCTCTCTGACTCTCAAATCTTT TAGTCATTATAGATcCCCCAGACTTTACATGACACAGCTTTATCACCAGAGAGGGACTGACACCCATGTTTCTCT~CCCC~G~AAAAT~CCAG~G~CTCTGATAGGCC~GT TTGTATCAGGTGCCCATCCC~G~GGTGCTGTTATCCAT~GG~G~ATATAT~GATGG~GCTTCCAGTCC~TCTCATGGAG~AGAAATACATA~TCC~G~G~ATG GGT~GTACTATTCTGATTACACAAAACAAATGCCACACATCACCCTTACCATGTGCCTGATCCAGCcTCTCCCCTGATTACACCA~CTCGTCTTCATT~CCTCTTCCATCATGTC CCCAAACCTGC~GGGCTCcCCACTGCCTACTGCATCGAGTCAAAACTCAAATGCTTGGCTTCTCATACGTCCACCAT~TCCTACC~TAGATTCCcCATTGCCTCCTCCTTCCC~ A~ACTCCACCCATCCTATCAGCCTGTCTCTTCCATATGACCTCATGCATCTCCACCTGCTCCCAGGCCAGT~GGGAAATAGAAAAACCC~CCCCCAAAT~G~GGGATGGATTCCA ACCCC~CTCCAGTAGC~G~ACAAATC~GCTTCAGTTTCCTGGTCTGTAG~GAGGGAT~GGTACCTTTCACATAGAGATCATCCTTTCCAGCAT~GG~CTAGCCACC~CTCT TGCAGGTCTC~CCCTT~GTCT~CTCTTAGACTTCTGCTTTCCACACCTGCACTGCTGTGCTGTGCCC~GTTGTGGTGCTGACAAA~TT~GAGCCTGCA~TGCCTT~CCGC G~CATAGCCCAGACACAG~GA~CTGGTTCTTACGATGGCACCCAGTGAGCACTCCC~GTCTACAGAGTGATAGCCTTCCGT~CCC~CTCTCCTGGACT~CTTG~TATCCCCT CCCAGTCACCTTGTGC~CCCTGCCCATCTGGGAAAATACCCCATCATTCATGCTACTGCC~CCTGGGGAGCCAGGGCTATGGGA~AGC~TTTTTTCCCCCCTAGAAACG~TGG ~C~TGTAAAACTTTAAAGCTCGAAAAC~TTGT~T~TGCTAAAGAAAAAGTCATCC~TCT~CCACATC~TA~GTCATTCCTGTATTCACCCGTCCAGACCTTGTTCACACTC TCACA~TTTAGAGTT~TCGT~TGTACAGATGGTTTTAT~TCTGATTTGTTTTCCTCTT~CGTTAGACCACAAATAGT~TCGCTTTCTATGTAGT~T~TTATCATT~A G~GACTCTACCAGAC~TATTCATTG~GTCAGATGTGGT~CTGTTAAATTGCTGTGTATCTGATAGCTCT~G~AGTCTATATGTTTGTAT~TGA~G~T~GTCATG TTCCTTC~GATCATGTACCCC~TTTACTTGCCATTACTC~TTGATAAACATTT~CTTGTTTCC~TGTTTA~AAATACATATTTTATAG~CTTCA~
1320 1440 1560 1680 1800 1920 2040 2160 2280 2400 2520 2640 2760 2880 3000 3115
960 291
Fig. 1. Sequence of V28 cDNA and deduced aa sequence. V28 cDNA was isolated ~ o m a human PBMC c D N A library by hybridization with a V28 genomic D N A clone, which in turn had been identified by PCR with degenerate primers as described (Schweickart et al., 1994). The position of the intron in the 5' U T R is indicated by an inverted triangle. Bars are drawn over the putative TM domains. The V28 GenBank accession No. is U20350 and the H G M W gene symbol is GPR13.
297 TABLE I G-protein-coupled receptor amina-acid identities (%) Protein
V28
MIPI~t/RANTES MCPR A MCPR B RBS 11 IL8R A ]L8R B EBII BLRI ATIIR 1 HM89
40 39 41 81 31 31 31 30 28 24
MIPI~/ RANTES
49 54 40 30 30 35 29 31 30
MCPR A
86 42 31 31 35 27 28 30
MCPR B
43 30 32 34 27 29 30
RBSll
33 29 32 30 26 29
The 3.1-kb V28 cDNA includes 1065 nt of coding sequence, 87 nt of 5'-UTR with an in-frame stop codon and 1960 nt of 3'-UTR including a poly(A) tail. The 1128 genomic clone contains the entire coding region on a 2.3-kb HindlII fragment. There are no introns within the coding region, as is common for chemokine receptor genes. The sequences of the genomic and cDNA clones diverge 10 nt upstream from the start ATG in the 5'-UTR, suggesting the presence of an intron in this position. The RBS11 genomic and cDNA sequences diverge at an equivalent position (Harrison et al., 1994). This is also similar to the human MIP-la/RANTES receptorencoding gene, which has an intron positioned at nt - 12 but no other introns withi~a the coding region (Gao et al.,
A
IL8R A
76 33 37 30 35
IL8R B
34 38 29 34
EBI1
33 28 32
BLR1
28 31
ATIIR 1
GenBank Accession No. L09230 U03882 U03905 U04808 M68932 M94582 L08176 X68149 M93394 D10924
32
1993). The human IL-8 receptor-encoding genes also contain introns only in their 5'-UTR (Kelvin et al., 1993).
(b) Expression of V28 examined by Northern blot The tissues expressing the highest level of 1,'28 RNA are brain, peripheral blood leukocytes (PBL) and spleen, as demonstrated by Northern blot (Fig. 2). Strong V28 expression was observed in several regions of the brain and other neural tissues, especially in medulla and spinal cord. A single transcript of approx. 3.2 kb correlated well with the length of the V28 cDNA clone. RNA from myeloid and lymphoid cell lines were also examined for V28 expression (Fig. 2B). 1128 was found to be expressed highly in THP.1 cells, at lower levels in U937 and HL60
oo~ ...
| B
~o~
THP.1p 013
u937p HL60p HL60d 013013 O13dayA
kb 9.5-kb
7.5--
9.5-7.5--
4.4--
4,.4--
2.4--
2.4t--
1.35.-
1.35--
Fig. 2. Tissue distribution of V28 by Northern blot. A 1.5-kb fragment of the V28 c D N A was labeled with 32p by the R a n d o m Primed D N A Labeling Kit (Boehringer-Mannheim) and used to probe Northern blots. (A) R N A from various h u m a n tissues was examined for the presence of V28 utilizing Northern blots from Clontech (Palo Alto, CA, USA) which contain approx. 2 Ixg poly(A)÷RNA from each tissue. Blots were probed and washed as per manufacturer's directions. S]~. muscle = skeletal muscle, Sin. int. = small intestine, Cereb. = cerebral cortex, Occip. pole = occipital pole. Standards in kb are indicated. (B) R N A from various h u m a n cell lines was also examined for V28. R N A was isolated from cell pellets using R N A STAT-60 (Tel-Test "B", Friendswood, TX, USA). 20 ~tg total R N A was loaded in each lane, separated on 2.2 M formaldehyde-l.2% agarose gels and transfered to nitrocellulose. Blots were probed in 50% formamide/5 × SSC/1 × Denhardt's/50 m M Na.phosphate, pH 6.5/100 ~tg per ml denatured salmon sperm D N A at 42°C and washed up to 50°C in 0.2 × SSC/0.1% SDS. Blots were reprobed with G A P D H as a control for R N A loading. Standards in kb are indicated. In the second panel, myeloid cells THP.1, U937 and HL60 were incubated with 50 ng/ml phorbol ester (p) or 1% D M S O (d) for the indicated number of days before harvesting for RNA.
298 cells, and not at all in lymphoid cell lines. THP.l, U937 and HL60 cells seemed to down-regulate V28 expression when induced to differentiate in culture with phorbol ester or DMSO, though the level of the control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript also appeared to decrease with differentiation. When Harrison et al. (1994) examined expression of RBSll in rat tissue RNA, high levels of expression of several transcript sizes were found in many regions of the brain. Significant expression was also observed in several other tissues, including kidney, small and large intestine, and lung, where there is little or no VZ8 expression.
26 25 24 23 22
P
21
(c) Mapping chromosomal location of V28 The V28 gene was mapped on human chromosomes using the entire cDNA as a probe on EcoRI-digested human-mouse somatic cell hybrid DNA (Naylor et al., 1983). V28 was positioned to chromosome 3, which had no discordant hybrids. One particular hybrid TSL-2, which has a chromosome 17/3 translocation (17pter-l7pl3::3p21-3pter) with no intact chromosome 3, scored positive for V28. This indicated that the V28 gene resides on chromosome 3p21-3pter (Fig. 3). This places it in close proximity to the related gene for MJP-laIRANTES receptor, which was mapped to 3~21 (Gao et al., 1993). (d) Possible function of V28 protein
We surmised that V28 might be a member of the chemokine receptor family based on its similarity to the MIP-laIRANTES and MCP receptors, its expression in leukocytes and the chromosomal location of its gene near the MIP-lol/RANTES receptor-encoding gene. In order to test this hypothesis, the V28 cDNA was stably expressed under control of the cytomegalovirus promoter in 293 cells and intracellular Ca2+ flux was measured after addition of various chemokines. The chemokines tested were MIP-la, MIP-lp, RANTES, MCP-1 and MCP-2 of the C-C chemokine family, and IL-8, neutrophi1 activating protein 2 (NAP-2), melanoma growth stimulating activity (MGSA), interferon-y-induced protein 10 (IP-lo), platelet factor 4 (PF-4) and ENAof the C-X-C chemokine family. However, no Ca2+ flux was detected in response to any of the peptides. As positive controls, the IL-8 receptor A and the MCP receptor B were expressed in the same manner in 293 cells and Ca2+ flux responses were observed after addition of IL-8 and MCP-1, respectively. V28-transfected 293 cells also failed to bind radiolabeled chemokines MCP-1, RANTES, MIP-la and MIP-IP. This limited search suggests that V28 does not interact with any of these known chemokines. It is interesting that V28 mRNA is found at such high
CHROMOSOME
3
Fig. 3. Chromosomal location of V28. The V28 gene was mapped to human chromosome 3p21-3pter by hybridization to human-mouse somatic cell hybrid DNA using the entire V28 cDNA as a probe. The positions of both 1128and the closely related MIPlol/RANTES receptor gene are indicated.
levels in brain and in lymphatic tissues. This distribution is similar to the human and murine orphan G-proteincoupled receptor BLRl (Dobner et al., 1992; Kaiser et al., 1993) and the closely related rat NLR (Kouba et al., 1993). There is a clear interconnection between the immune and nervous systems. Many neuropeptides have been localized to nerve endings innervating lymphoid organs (Weihe et al., 1991). Neuropeptides such as neuropeptide Y and vasoactive intestinal peptide have been shown to affect leukocyte migration (Ottaway and Husband, 1992). Identification of a V28 ligand may contribute to our understanding of the interactions between the nervous and immune systems.
ACKNOWLEDGEMENTS
We thank Larry Tjoelker for the PBMC cDNA library, David Chantry for RNA samples, Ron Godiska for initial
299 cloning experiments, Dina Leviten and Christi Wood for sequencing and oligodeoxynucleotide synthesis.
REFERENCES Charo, I.F., Myers, S.J., Hermala, A., Franci, C., Connolly, A.J. and Coughlin, S.R.: Molecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the C-terminal tails. Proc. Natl. Acad. Sci. USA 91 (1994) 2752-2756. Dobner, T., Wolf, I., Emrich, T. and Lipp, M.: Differentiation-specific expression of a novel G protein-coupled receptor from Burkitt's lymphoma. Eur. J. Immunol. 22 (1992) 2795-2799. Gao, J.-L., Kuhns, D.B., Tiffany, H.L., McDermott, D., Li, X., Francke, U. and Murphy, P.M.: Structure and functional expression of the human macrophage inflammatory protein lc~/RANTES receptor. J. Exp. Med. 177 (1993) 1421-1427. Gilman, A.G.: G proteins: tran:~ducers of receptor-generated signals. Annu. Rev. Biochem. 56 (19~;7) 615-629. Harrison, J.K., Barber, C.M. and Lynch, K.R.: cDNA cloning of a G-protein-coupled receptor e,xpressed in rat spinal cord and brain related to chemokine receptcrs. Neurosci. Lett. 169 (1994) 85-89. Holmes, W.E., Lee, J., Kuang, W.-J., Rice, G.C. and Wood, W.I.: Structure and functional expression of a human inter!eukin-8 receptor. Science 253 (1991) 1278--1280. Kaiser, E., F0rster, R., Wolf, I., Ebensperger, C., Kuehl, W.M. and Lipp, M.: The G protein-coupled receptor BLR1 is involved in murine B cell differentiation and is also expressed in neuronal tissues. Eur. J. Immunol. 23 (1993) 2532-2539.
Kelvin, D.J., Michiel, D.F., Johnston, J.A., Lloyd, A.R., Sprenger, H., Oppenheim, J.J. and Wang, J.-M.: Chemokines and serpentines: the molecular biology of chemokine receptors. J. Leukoc. Biol. 54 (1993) 604-612. Kouba, M., Vanetti, M., Wang, X., Sch~ifer, M. and H011t, V.: Cloning of a novel putative G-protein-coupled receptor (NLR) which is expressed in neuronal and lymphatic tissues. FEBS Lett. 321 (1993) 173-178. Murphy, P.M. and Tiffany, H.L.: Cloning of complementary DNA encoding a functional human interleukin-8 receptor. Science 253 (1991) 1280-1283. Naylor, S.L., Sakaguchi, A.Y., Shows, T.B., Law, M.L., Goeddel, D.V. and Gray, P.W.: Human immune interferon gene is located on chromosome 12. J. Exp. Med. 57 (1983) 1020-1027. Neote, K., DiGregorio, D., Mak, J.Y., Horuk, R. and Schall, T.J.: Molecular cloning, functional expression, and signaling characteristics of a C-C chemokine receptor. Cell 72 (1993) 415-425. Oppenheim, J.J., Zachariae, C.O.C., Mukaida, N. and Matsushima, K.: Properties of the novel proinflammatory supergene "intercrine" cytokine family. Annu. Rev. Immunol. 9 (1991) 617-648. Ottaway, C.A. and Husband, A.J.: Central nervous system influences on lymphocyte migration. Brain Behav. Immun. 6 (1992) 97-116. Probst, W.C., Snyder, L.A. Schuster, D.I., Brosius, J. and Sealfon, S.C.: Sequence alignment of the G-protein coupled receptor superfamily. DNA Cell Biol. 11 (1992) 1-20. Schweickart, V.L., Raport, C.J., Godiska, R., Byers, M.G., Eddy Jr., R.L., Shows, T.B. and Gray, P.W.: Cloning of human and mouse EBI1, a lymphoid-specific G-protein-coupled receptor encoded on human chromosome 17q12-q21.2. Genomics 23 (1994) 643-650. Weihe, E., Nohr, D., Michel, S., Muller, S., Zentel, H.-J., Fink, T. and Krekel, J.: Molecular anatomy of the neuro-immune connection. Int. J. Neurosci. 59 (1991) 1-23.
295
GENE 09054
The orphan G-protein-coupled receptor-encoding gene V28 is closely related to genes for chemokine receptors and is expressed in lymphoid and neural tissues (Degenerate primer PCR; inflammation; human genomic DNA)
Carol J. Raport a, Vicki L. Schweickart", Roger L. Eddy Jr. b, Thomas B. S h o w s b and Patrick W. Gray a alCOS Corporation, Bothell, WA 98021, USA; and bDepartment of Human Genetics, Roswell Park Cancer Institute, Buffalo, NY14263, USA. Tel. (1-716) 845-3108
Received by A.D. Riggs: 23 January 1995; Revised/Accepted: 29 March/30 March 1995; Received at publishers: 15 May 1995
SUMMARY
A polymerase chain reaction (PCR) strategy with degenerate primers was used to identify novel G-protein-coupled receptor-encoding genes from human genomic DNA. One of the isolated clones, termed V28, showed high sequence similarity to the genes encoding human chemokine receptors for monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1~ (MIP-I~)/RANTES, and to the rat orphan receptor-encoding gene RBS11. When RNA was analyzed by Northern blot, V28 was found to be most highly expressed in neural and lymphoid tissues. Myeloid cell lines, particularly THP.1 cells, showed especially high expression of V28. We have mapped V28 to human chromosome 3p21-3pter, near the MIP-I~/RANTES receptor-encoding gene.
INTRODUCTION
G-protein-coupled receptors (GCRs) are cell-surface receptors which recognize extracellular signals and transduce those signals into an intracellular response (Gilman, Correspondence to: Dr. P.W. Gray, ICOS Corporation, 22021 20th Avenue S.E., Bothell, WA 980:21, USA. Tel. (1-206) 485-1900; Fax (1-206) 485-1961; e-mail: pgray~icos.com
Abbreviations: aa, amino acid(s); ATIIR 1, angiotensin II receptor type 1; BLR1, Burkitt's lymphoma receptor 1; bp, base pair(s); DMSO, dimethylsulfoxide; EBI1, Epstein-Barr-induced receptor 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GCR, G protein-coupled receptor; HGMW, Human Gene Mapping Workshop; IL, interleukin; kb, kilobase(s) or 1000 lap; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; NLR, neurolymphatic receptor; nt, nucleotide(s); PBMC, peripheral blood mononuclear cell; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaC1/0.0t5 M Na3"citrate pH 7.6; TM, transmembrane; UTR, untranslated region(s); V28, gene encoding orphan GCR; X, any aa. SSDI 0378-1119(95)00336-3
1987). The signals that act through GCRs are quite diverse, ranging from photons of light to catecholamines to peptides. The receptors themselves share striking structural similarity, containing seven hydrophobic ~-helical membrane-spanning domains with an extracellular Nterminus. Ligand recognition by the receptor leads to G-protein activation and to intracellular responses which include altered cyclic nucleotide metabolism, phospholipid hydrolysis, intracellular ion fluxes and changes in surface ion-channel activity. G-protein-coupled receptors are key regulators of many immune and homeostatic responses. Chemokines, a family of pro-inflammatory peptides, promote chemotaxis in leukocytes by binding to GCRs (Oppenheim et al., 1991). The cloning of two receptor-encoding genes for interleukin 8 (IL-8) (Holmes et al., 1991; Murphy and Tiffany, 1991) first demonstrated that chemokine receptors are members of the GCR family. Subsequently, degenerate PCR techniques were used to clone two addi-
296 tional genes encoding GCRs which recognize the chemokines MIP-I~/RANTES (Neote et al., 1993) and MCP-1 (Charo et al., 1994). We also have utilized a degenerate PCR strategy to identify additional members of the human GCR family which may be involved in immune function. We describe here one such receptor-encoding gene which we called V28.
EXPERIMENTAL AND DISCUSSION
(a) Cloning of the 1/28 gene Human genomic DNA was screened by PCR using degenerate primers to identify genes related to the IL-8 receptors as described by Schweickart et al. (1994). Seven unique PCR fragments were obtained and used to screen a human genomic DNA library (Stratagene) and a human peripheral blood mononuclear cell (PBMC) cDNA plas-
mid library as described by Schweickart et al. (1994). One gene identified in this manner was termed V28. As shown in Fig. 1, the deduced aa sequence of V28 reveals a 355-aa polypeptide which displays the features of other GCRs, including seven hydrophobic domains which may function as TM regions. The V28 protein sequence also contains approx. 20 aa that are highly conserved among members of the GCR superfamily (Probst et al., 1992), though there is no concensus sequence for N-linked glycosylation in the extracellular regions. The V28 sequence includes an aa motif (DRYLAIV) in the second cytoplasmic loop which is particularly conserved among chemokine receptors. V28 shows highest sequence similarity to the rat orphan GCR-encoding gene RBSI I (Harrison et al., 1994), 79% identical at the nt level and 81% identical at the aa level, and to human chemokine receptors for MIP-I~/RANTES and for MCP-1 (see Table I).
V, ACT~GTCTCTGGTAAAGTCTGAGcAGGACAGGGTGGCTGACTGGCAGATCCAGAGGTTCCCTTGGCAGTCCACGCCAGGCCTTCACCATGGATCAGTTCCCTG~TCAGTGACAGAAAAC M D Q F P E S V T E N
120 II
TTT~GTACGATGATT~GCTGAGGCCTGTTATATTGG~ACATCGTGGTCTTT~GACTGTGTTCCTGTCCATATTCTACTCCGTCATCTTTGCCA~CCT~AAATTTGTTG F E Y D D L A E A C Y I G D I V V F G T V F L S I F Y S V I F A I G L V G N L L
240 51
GTAGTGTTTGCCCTCACC~CAGC~G~GCCC~GAGTGTCACCGACATTTACCTCCTG~CC~GCCTTGTCTGATCTGCTGTTTGTAGCCACTT~CCC~CT~ACTCACTATTTG V V F A L T N S K K P K S V T D I Y L L N L A L S D L L F V A T L P F W T H Y L
360 91
TM3 ATAAATGAAAAGGGCCTCCAC~TGCCATGTGCAAATTCACTACCGCCTTCTTCTTCATCGGCTTTTTT~GCATATTCTTCATCACCGTCATCAGCATTGATA~TACCTGGCCATC I N E K G L H N A M C K F T T A F F F I G F F G S I F F I T V I S I D R Y L A I TM4 GTCCT~CCGCC~CTCCATG~C~CCGGACCGTGCAGCATGGCGTCACCATCAGCCTAGGCGTCTGGGCAGCAGCCATTTTGGT~CAGCACCCCAGTTCATGTTCACAAAGCAGAAA V L A A N S M N N R T V Q H G V T I S L G V W A A A I L V A A P Q F M F T K Q K TM~ GAAAATG~TGCCT~GTGACTACCCCGAGGTCCTCCA~AAATCTGGCCCGTGCTCCGC~TGTGGAAACAAATTTTCTTGGCTTcCTACTCCCCCTGCTCATTATGAGTTATTGCTAC E N E C L G D Y P E V L Q E I W P V L R N V E T N F L G F L L P L L I M S Y C Y
480 131 600 171 720 211
TM~ TTCAG~TCATCCAGACGCTGTTTTCCTGC~G~CCAC~GAAAGCCAAAGCCATTAAACTGATCCTTCTGGTGGTCATCGTGTTTTTCCTCTTCTGGACACCCTAC~CGTTATGATT F R I I Q T L F S C K N H K K A K A I K L I L L V V I V F F L F W T P Y N V M I TM7 TTCCTGGAGACGCTT~GCTCTATGACTTCTTTCCCAGTTGTGACATGA~ATCTGAGGCTGGCCCTCAGTGTGACTGAGAC~TTGCATTTAGCCATTGTTGCCTG~TCCTCTC F L E T L K L Y D F F P S C D M R K D L R L A L S V T E T V A F S H C C L N P L
840 251
ATCTATGCATTT~T~GGAG~GTTCAG~GATACCTTTACCACCTGTATGGGAAATGCCTGGCTGTCCTGTGT~GCGCTCAGTCCACGTTGATTTCTCCTCATCTG~TCACAAAGG I Y A F A G E K F R R Y L Y H L Y G K C L A V L C G R S V H V D F S S S E S Q R
1080 331
A~AG~ATGG~GTGTTCTGAGCAGC~TTTTACTTACCACACGAGTGATGGAGATGCATTGCTCCTTCTCTG~GGG~TCCCAAAGCCTTGTGTCTACAGAG~CCT~AGTTCCTG S R H G S V L S S N F T Y H T S D G D A L L L L
1200 355
~CCT~TGCTGACTAGTGAGGAAAGATTTTTGTTGTTATTTCTTACAGGCACAAAATGATGGACCC~TGCACACAAAAC~CCCTAGAGTGTTGTTGAG~TTGTGCTCAAAA~TGA AG~TG~CAAATTG~C~TTTG~TGACAAAGAGTAGACATTTCTCTTACTGCAAATGTCATCAG~CTTTTTGGTTTGCAGATGACAAAAATTC~CTCAGACTAGTTTAGTTAAAT GAG~GTG~TATTGTTCATATTGTGGCAC~GCAAAA~GTGTCTGAGCCCTCAAAGTGAGGGGAAACCA~GCC~AGCC~GCTAG~TTCCCTCTCTCTGACTCTCAAATCTTT TAGTCATTATAGATcCCCCAGACTTTACATGACACAGCTTTATCACCAGAGAGGGACTGACACCCATGTTTCTCT~CCCC~G~AAAAT~CCAG~G~CTCTGATAGGCC~GT TTGTATCAGGTGCCCATCCC~G~GGTGCTGTTATCCAT~GG~G~ATATAT~GATGG~GCTTCCAGTCC~TCTCATGGAG~AGAAATACATA~TCC~G~G~ATG GGT~GTACTATTCTGATTACACAAAACAAATGCCACACATCACCCTTACCATGTGCCTGATCCAGCcTCTCCCCTGATTACACCA~CTCGTCTTCATT~CCTCTTCCATCATGTC CCCAAACCTGC~GGGCTCcCCACTGCCTACTGCATCGAGTCAAAACTCAAATGCTTGGCTTCTCATACGTCCACCAT~TCCTACC~TAGATTCCcCATTGCCTCCTCCTTCCC~ A~ACTCCACCCATCCTATCAGCCTGTCTCTTCCATATGACCTCATGCATCTCCACCTGCTCCCAGGCCAGT~GGGAAATAGAAAAACCC~CCCCCAAAT~G~GGGATGGATTCCA ACCCC~CTCCAGTAGC~G~ACAAATC~GCTTCAGTTTCCTGGTCTGTAG~GAGGGAT~GGTACCTTTCACATAGAGATCATCCTTTCCAGCAT~GG~CTAGCCACC~CTCT TGCAGGTCTC~CCCTT~GTCT~CTCTTAGACTTCTGCTTTCCACACCTGCACTGCTGTGCTGTGCCC~GTTGTGGTGCTGACAAA~TT~GAGCCTGCA~TGCCTT~CCGC G~CATAGCCCAGACACAG~GA~CTGGTTCTTACGATGGCACCCAGTGAGCACTCCC~GTCTACAGAGTGATAGCCTTCCGT~CCC~CTCTCCTGGACT~CTTG~TATCCCCT CCCAGTCACCTTGTGC~CCCTGCCCATCTGGGAAAATACCCCATCATTCATGCTACTGCC~CCTGGGGAGCCAGGGCTATGGGA~AGC~TTTTTTCCCCCCTAGAAACG~TGG ~C~TGTAAAACTTTAAAGCTCGAAAAC~TTGT~T~TGCTAAAGAAAAAGTCATCC~TCT~CCACATC~TA~GTCATTCCTGTATTCACCCGTCCAGACCTTGTTCACACTC TCACA~TTTAGAGTT~TCGT~TGTACAGATGGTTTTAT~TCTGATTTGTTTTCCTCTT~CGTTAGACCACAAATAGT~TCGCTTTCTATGTAGT~T~TTATCATT~A G~GACTCTACCAGAC~TATTCATTG~GTCAGATGTGGT~CTGTTAAATTGCTGTGTATCTGATAGCTCT~G~AGTCTATATGTTTGTAT~TGA~G~T~GTCATG TTCCTTC~GATCATGTACCCC~TTTACTTGCCATTACTC~TTGATAAACATTT~CTTGTTTCC~TGTTTA~AAATACATATTTTATAG~CTTCA~
1320 1440 1560 1680 1800 1920 2040 2160 2280 2400 2520 2640 2760 2880 3000 3115
960 291
Fig. 1. Sequence of V28 cDNA and deduced aa sequence. V28 cDNA was isolated ~ o m a human PBMC c D N A library by hybridization with a V28 genomic D N A clone, which in turn had been identified by PCR with degenerate primers as described (Schweickart et al., 1994). The position of the intron in the 5' U T R is indicated by an inverted triangle. Bars are drawn over the putative TM domains. The V28 GenBank accession No. is U20350 and the H G M W gene symbol is GPR13.
297 TABLE I G-protein-coupled receptor amina-acid identities (%) Protein
V28
MIPI~t/RANTES MCPR A MCPR B RBS 11 IL8R A ]L8R B EBII BLRI ATIIR 1 HM89
40 39 41 81 31 31 31 30 28 24
MIPI~/ RANTES
49 54 40 30 30 35 29 31 30
MCPR A
86 42 31 31 35 27 28 30
MCPR B
43 30 32 34 27 29 30
RBSll
33 29 32 30 26 29
The 3.1-kb V28 cDNA includes 1065 nt of coding sequence, 87 nt of 5'-UTR with an in-frame stop codon and 1960 nt of 3'-UTR including a poly(A) tail. The 1128 genomic clone contains the entire coding region on a 2.3-kb HindlII fragment. There are no introns within the coding region, as is common for chemokine receptor genes. The sequences of the genomic and cDNA clones diverge 10 nt upstream from the start ATG in the 5'-UTR, suggesting the presence of an intron in this position. The RBS11 genomic and cDNA sequences diverge at an equivalent position (Harrison et al., 1994). This is also similar to the human MIP-la/RANTES receptorencoding gene, which has an intron positioned at nt - 12 but no other introns withi~a the coding region (Gao et al.,
A
IL8R A
76 33 37 30 35
IL8R B
34 38 29 34
EBI1
33 28 32
BLR1
28 31
ATIIR 1
GenBank Accession No. L09230 U03882 U03905 U04808 M68932 M94582 L08176 X68149 M93394 D10924
32
1993). The human IL-8 receptor-encoding genes also contain introns only in their 5'-UTR (Kelvin et al., 1993).
(b) Expression of V28 examined by Northern blot The tissues expressing the highest level of 1,'28 RNA are brain, peripheral blood leukocytes (PBL) and spleen, as demonstrated by Northern blot (Fig. 2). Strong V28 expression was observed in several regions of the brain and other neural tissues, especially in medulla and spinal cord. A single transcript of approx. 3.2 kb correlated well with the length of the V28 cDNA clone. RNA from myeloid and lymphoid cell lines were also examined for V28 expression (Fig. 2B). 1128 was found to be expressed highly in THP.1 cells, at lower levels in U937 and HL60
oo~ ...
| B
~o~
THP.1p 013
u937p HL60p HL60d 013013 O13dayA
kb 9.5-kb
7.5--
9.5-7.5--
4.4--
4,.4--
2.4--
2.4t--
1.35.-
1.35--
Fig. 2. Tissue distribution of V28 by Northern blot. A 1.5-kb fragment of the V28 c D N A was labeled with 32p by the R a n d o m Primed D N A Labeling Kit (Boehringer-Mannheim) and used to probe Northern blots. (A) R N A from various h u m a n tissues was examined for the presence of V28 utilizing Northern blots from Clontech (Palo Alto, CA, USA) which contain approx. 2 Ixg poly(A)÷RNA from each tissue. Blots were probed and washed as per manufacturer's directions. S]~. muscle = skeletal muscle, Sin. int. = small intestine, Cereb. = cerebral cortex, Occip. pole = occipital pole. Standards in kb are indicated. (B) R N A from various h u m a n cell lines was also examined for V28. R N A was isolated from cell pellets using R N A STAT-60 (Tel-Test "B", Friendswood, TX, USA). 20 ~tg total R N A was loaded in each lane, separated on 2.2 M formaldehyde-l.2% agarose gels and transfered to nitrocellulose. Blots were probed in 50% formamide/5 × SSC/1 × Denhardt's/50 m M Na.phosphate, pH 6.5/100 ~tg per ml denatured salmon sperm D N A at 42°C and washed up to 50°C in 0.2 × SSC/0.1% SDS. Blots were reprobed with G A P D H as a control for R N A loading. Standards in kb are indicated. In the second panel, myeloid cells THP.1, U937 and HL60 were incubated with 50 ng/ml phorbol ester (p) or 1% D M S O (d) for the indicated number of days before harvesting for RNA.
298 cells, and not at all in lymphoid cell lines. THP.l, U937 and HL60 cells seemed to down-regulate V28 expression when induced to differentiate in culture with phorbol ester or DMSO, though the level of the control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript also appeared to decrease with differentiation. When Harrison et al. (1994) examined expression of RBSll in rat tissue RNA, high levels of expression of several transcript sizes were found in many regions of the brain. Significant expression was also observed in several other tissues, including kidney, small and large intestine, and lung, where there is little or no VZ8 expression.
26 25 24 23 22
P
21
(c) Mapping chromosomal location of V28 The V28 gene was mapped on human chromosomes using the entire cDNA as a probe on EcoRI-digested human-mouse somatic cell hybrid DNA (Naylor et al., 1983). V28 was positioned to chromosome 3, which had no discordant hybrids. One particular hybrid TSL-2, which has a chromosome 17/3 translocation (17pter-l7pl3::3p21-3pter) with no intact chromosome 3, scored positive for V28. This indicated that the V28 gene resides on chromosome 3p21-3pter (Fig. 3). This places it in close proximity to the related gene for MJP-laIRANTES receptor, which was mapped to 3~21 (Gao et al., 1993). (d) Possible function of V28 protein
We surmised that V28 might be a member of the chemokine receptor family based on its similarity to the MIP-laIRANTES and MCP receptors, its expression in leukocytes and the chromosomal location of its gene near the MIP-lol/RANTES receptor-encoding gene. In order to test this hypothesis, the V28 cDNA was stably expressed under control of the cytomegalovirus promoter in 293 cells and intracellular Ca2+ flux was measured after addition of various chemokines. The chemokines tested were MIP-la, MIP-lp, RANTES, MCP-1 and MCP-2 of the C-C chemokine family, and IL-8, neutrophi1 activating protein 2 (NAP-2), melanoma growth stimulating activity (MGSA), interferon-y-induced protein 10 (IP-lo), platelet factor 4 (PF-4) and ENAof the C-X-C chemokine family. However, no Ca2+ flux was detected in response to any of the peptides. As positive controls, the IL-8 receptor A and the MCP receptor B were expressed in the same manner in 293 cells and Ca2+ flux responses were observed after addition of IL-8 and MCP-1, respectively. V28-transfected 293 cells also failed to bind radiolabeled chemokines MCP-1, RANTES, MIP-la and MIP-IP. This limited search suggests that V28 does not interact with any of these known chemokines. It is interesting that V28 mRNA is found at such high
CHROMOSOME
3
Fig. 3. Chromosomal location of V28. The V28 gene was mapped to human chromosome 3p21-3pter by hybridization to human-mouse somatic cell hybrid DNA using the entire V28 cDNA as a probe. The positions of both 1128and the closely related MIPlol/RANTES receptor gene are indicated.
levels in brain and in lymphatic tissues. This distribution is similar to the human and murine orphan G-proteincoupled receptor BLRl (Dobner et al., 1992; Kaiser et al., 1993) and the closely related rat NLR (Kouba et al., 1993). There is a clear interconnection between the immune and nervous systems. Many neuropeptides have been localized to nerve endings innervating lymphoid organs (Weihe et al., 1991). Neuropeptides such as neuropeptide Y and vasoactive intestinal peptide have been shown to affect leukocyte migration (Ottaway and Husband, 1992). Identification of a V28 ligand may contribute to our understanding of the interactions between the nervous and immune systems.
ACKNOWLEDGEMENTS
We thank Larry Tjoelker for the PBMC cDNA library, David Chantry for RNA samples, Ron Godiska for initial
299 cloning experiments, Dina Leviten and Christi Wood for sequencing and oligodeoxynucleotide synthesis.
REFERENCES Charo, I.F., Myers, S.J., Hermala, A., Franci, C., Connolly, A.J. and Coughlin, S.R.: Molecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the C-terminal tails. Proc. Natl. Acad. Sci. USA 91 (1994) 2752-2756. Dobner, T., Wolf, I., Emrich, T. and Lipp, M.: Differentiation-specific expression of a novel G protein-coupled receptor from Burkitt's lymphoma. Eur. J. Immunol. 22 (1992) 2795-2799. Gao, J.-L., Kuhns, D.B., Tiffany, H.L., McDermott, D., Li, X., Francke, U. and Murphy, P.M.: Structure and functional expression of the human macrophage inflammatory protein lc~/RANTES receptor. J. Exp. Med. 177 (1993) 1421-1427. Gilman, A.G.: G proteins: tran:~ducers of receptor-generated signals. Annu. Rev. Biochem. 56 (19~;7) 615-629. Harrison, J.K., Barber, C.M. and Lynch, K.R.: cDNA cloning of a G-protein-coupled receptor e,xpressed in rat spinal cord and brain related to chemokine receptcrs. Neurosci. Lett. 169 (1994) 85-89. Holmes, W.E., Lee, J., Kuang, W.-J., Rice, G.C. and Wood, W.I.: Structure and functional expression of a human inter!eukin-8 receptor. Science 253 (1991) 1278--1280. Kaiser, E., F0rster, R., Wolf, I., Ebensperger, C., Kuehl, W.M. and Lipp, M.: The G protein-coupled receptor BLR1 is involved in murine B cell differentiation and is also expressed in neuronal tissues. Eur. J. Immunol. 23 (1993) 2532-2539.
Kelvin, D.J., Michiel, D.F., Johnston, J.A., Lloyd, A.R., Sprenger, H., Oppenheim, J.J. and Wang, J.-M.: Chemokines and serpentines: the molecular biology of chemokine receptors. J. Leukoc. Biol. 54 (1993) 604-612. Kouba, M., Vanetti, M., Wang, X., Sch~ifer, M. and H011t, V.: Cloning of a novel putative G-protein-coupled receptor (NLR) which is expressed in neuronal and lymphatic tissues. FEBS Lett. 321 (1993) 173-178. Murphy, P.M. and Tiffany, H.L.: Cloning of complementary DNA encoding a functional human interleukin-8 receptor. Science 253 (1991) 1280-1283. Naylor, S.L., Sakaguchi, A.Y., Shows, T.B., Law, M.L., Goeddel, D.V. and Gray, P.W.: Human immune interferon gene is located on chromosome 12. J. Exp. Med. 57 (1983) 1020-1027. Neote, K., DiGregorio, D., Mak, J.Y., Horuk, R. and Schall, T.J.: Molecular cloning, functional expression, and signaling characteristics of a C-C chemokine receptor. Cell 72 (1993) 415-425. Oppenheim, J.J., Zachariae, C.O.C., Mukaida, N. and Matsushima, K.: Properties of the novel proinflammatory supergene "intercrine" cytokine family. Annu. Rev. Immunol. 9 (1991) 617-648. Ottaway, C.A. and Husband, A.J.: Central nervous system influences on lymphocyte migration. Brain Behav. Immun. 6 (1992) 97-116. Probst, W.C., Snyder, L.A. Schuster, D.I., Brosius, J. and Sealfon, S.C.: Sequence alignment of the G-protein coupled receptor superfamily. DNA Cell Biol. 11 (1992) 1-20. Schweickart, V.L., Raport, C.J., Godiska, R., Byers, M.G., Eddy Jr., R.L., Shows, T.B. and Gray, P.W.: Cloning of human and mouse EBI1, a lymphoid-specific G-protein-coupled receptor encoded on human chromosome 17q12-q21.2. Genomics 23 (1994) 643-650. Weihe, E., Nohr, D., Michel, S., Muller, S., Zentel, H.-J., Fink, T. and Krekel, J.: Molecular anatomy of the neuro-immune connection. Int. J. Neurosci. 59 (1991) 1-23.