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(Journal of Leukocyte Biology. 2001;69:297-305.)
© 2001 by Society for Leukocyte Biology

Cloning, mRNA distribution, and functional expression of an avian counterpart of the chemokine receptor/HIV coreceptor CXCR4

Thomas S. Liang, Jennifer K. Hartt, Shuyan Lu*, Manuela Martins-Green*, Ji-Liang Gao and Philip M. Murphy

Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland; and
* Department of Cell Biology and Neurosciences, University of California, Riverside

Correspondence: Philip M. Murphy, M.D., Laboratory of Host Defenses, NIAID, Building 10, Room 11N113, National Institutes of Health, Bethesda, MD 20892. E-mail: pmm{at}nih.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The chemokine signaling system, which coordinates the basal and emergency trafficking of leukocytes, presumably coevolved with the hematopoietic system. To study its phylogenetic origins, we used the open reading frame (ORF) of the human chemokine receptor CXCR4 as a genomic probe, since in mammals it is the most highly conserved chemokine receptor known. CXCR4 cross-hybridized to genomic DNA from mouse and chicken, but not zebrafish, Drosophila, or Caenorhabditis elegans. Accordingly, we cloned the corresponding chicken cDNA. The ORF is 359 codons long versus 352 for human CXCR4, and encodes a protein 82% identical to human CXCR4. In a calcium flux assay of receptor function, CHO-K1 cells stably transfected with the chicken cDNA responded specifically to human SDF-1, the specific ligand for CXCR4, but not to a panel of other chemokines tested at 100 nM. SDF-1 activated the cells in a dose-dependent manner (EC50 ~5 nM), whereas parental CHO-K1 cells did not respond. The CHO-K1 cell transfectants also bound 125I-SDF-1 specifically. Leukocytes from chicken peripheral blood expressed chCXCR4 mRNA and responded to human SDF-1 in a calcium flux assay with an EC50 similar to that for chCXCR4-transfected CHO cells, suggesting that this response is mediated by native chCXCR4. Analysis of chicken genomic DNA with the chicken cDNA as probe revealed a pattern consistent with a single copy gene, and the absence of any closely related genes. mRNA was detected in brain, bursa, liver, small and large intestine, embryonal fibroblasts, and blood leukocytes, but not in stomach or pancreas. These results, which identify the first functional non-viral, non-mammalian chemokine receptor, suggest that the origins of a functional chemokine system extend at least to birds and suggest that, as in mammals, CXCR4 functions in many avian tissues.

Key Words: G protein-coupled receptor • SDF-1 • inflammation • evolution • development • signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The chemokine signaling system, which consists of at least 43 extracellular protein ligands and 18 G protein-coupled receptors, orchestrates specific leukocyte trafficking in mammals and regulates innate and adaptive immunity, pathological inflammation, HIV infection, and development, among other processes [1 2 3 4 5 6 7 8 ]. Because the chemokine system exhibits differential specificity for leukocyte subtypes, it is reasonable to hypothesize that it coevolved with the hematopoietic system. Furthermore, like any receptor-mediated signaling system, chemokine ligands must necessarily have coevolved with their receptors, although they could have arisen at different times. To date chemokine-like sequences have been reported in bird (Gallus gallus), fish, and lamprey, and chemokine receptor-like sequences have been reported in bird (G. gallus), frog (Xenopus laevis) and fish (Cyprinus carpio and Oncorhynchus mykiss) [9 10 11 12 13 14 15 ]. However, functional chemokine-chemokine receptor signaling has been identified only in mammals.

One strategy for tracing the origins of genes is iterative cross-hybridization, however, in general the chemokine system is poorly suited for this because it is evolving at an unusually rapid rate. For example, human-mouse chemokine or chemokine receptor orthologs generally range from 25–50% divergence in amino acid sequence [1 ]. One exception is the CXC chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4, which are ~2 and 6% divergent between human and mouse, respectively [1 ]. CXCR4, unlike other chemokine receptors, is widely expressed in the adult mammal (brain, thymus, lymph node, spleen, bone marrow, stomach, small intestine, liver, and kidney), as well as throughout embryonic development [16 17 18 19 20 ]. Consistent with this, CXCR4 is involved in hematopoiesis, gastric vasculogenesis, and development of the heart and cerebellum, as revealed by the phenotype of CXCR4-/- mice [21 22 23 24 ]. Moreover, human CXCR4 is exploited by HIV as a cell entry factor [19 , 25 ]. SDF-1 and CXCR4 comprise a monogamous signaling unit in mammals and are essential genes, unlike other chemokines and chemokine receptors studied so far [1 ].

CXCR4 may be the gene most closely linked to the ancestral chemokine receptor. Because it is so highly conserved in mammals we hypothesized that it may be possible to probe the origins of the chemokine system through the use of iterative cross-hybridization with CXCR4 probes. Moreover, because SDF-1 is so highly conserved, we hypothesized that it may be possible to use human SDF-1 as a cross-species agonist to identify ancient, functional CXCR4 orthologs. Here we present the first result of this approach.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
Parental CHO-K1 cells were obtained from ATCC (Manassas, VA). Freshly isolated chicken blood was obtained from Dan Pare (National Institute of Allergy and Infectious Diseases, National Institutes of Health). Ten milliliters of chicken blood was washed and resuspended in 40 mL of phosphate-buffered saline (PBS). Ten milliliters of lymphocyte separation medium was added slowly to the bottom, and the cells were centrifuged for 30 min at 1600 rpm. The layer of chicken leukocytes was collected between the first two phases. Cells were then washed once in PBS.

Isolation of genomic DNA
Genomic DNA from chicken and drosophila was obtained from Clontech (Palo Alto, CA). Zebrafish (D. rerio) genomic DNA was isolated from whole fish (gift of Brant Weinstein, National Institutes of Health) by overnight digestion at 55°C in lysis buffer [100 mM Tris-HCl, pH ~8.0, 5 mM EDTA, 0.2% sodium dodecyl sulfate (SDS), 200 mM NaCl] and proteinase K. C. elegans genomic DNA was a generous gift from Phil Morgan (Case Western Reserve University). Human genomic DNA was a gift from Dave McDermott (National Institutes of Health).

Analysis of genomic DNA
Five micrograms of genomic DNA was digested with a restriction enzyme (EcoRI, XbaI, or PstI) overnight at 37°C, and fragments were separated by size by electrophoresis through a 0.8% agarose gel. A Southern blot was then prepared by standard methods and was prehybridized in Hybrisol I (Oncor, Gaithersburg, MD) for 20 min at 37°C and hybridized overnight at 37°C with a CXCR4 ORF probe (~1.5 million specific counts/mL), which was labeled with [32P]dCTP using a random primer-labeling kit (Boehringer Mannheim, Indianapolis, IN). Blots of chicken genomic DNA were washed at high stringency (0.1x SSPE/0.1% SDS for 20 min at 65°C) and exposed overnight. Zooblots were washed at low stringency (5x SSPE/0.1% SDS for 20 min at 45°C) and exposed for 1–5 days.

DNA cloning
We amplified by polymerase chain reaction (PCR) a 392-bp fragment from chicken genomic DNA with the use of the following degenerate primers, which were designed based on human and mouse CXCR4 nucleotide sequences: sense, 5’-GC[AT]GT[TC]CAT[GA]TCAT[TC]TACAC[AT]GTCAACCTCTA-3’, and antisense, 5’GT[GC]GTCTT[GC]A[AG]GGC[TC]TTGCGCTTCTGGTGGCC-3’. The PCR protocol was 5 min at 94°C, 35 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 70°C, followed by a final extension time of 10 min at 72°C. Amplification was performed in a 50 µL PCR tube containing 5 µL of genomic DNA (0.1 µg/µL), 0.5 µg of each primer, 10 mM dNTP, 1.25 U of DNA Platinum Taq Polymerase, 25 mM of MgSO4, in PCR buffer (Life Technologies). PCR products were purified by agarose gel electrophoresis, cloned into plasmid pCR2.1 with a TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced on both strands. The 5’ and 3’ ends of the ORF were obtained by PCR amplification from a chicken liver cDNA library with internal and vector primers. The chicken CXCR4 primers were 5’-ACATTTCCGAAGTACCAGCTTATGGCTGCA-3’ and 5’-TCCACAGACCAGAATGGCAAGGTGATGACA-3’, and Lambda gt11 primers were 5’-GGGATTGGTGGCGACGACTCCTGG-3’ and 5’-CCAACTGGTAATGGTAGCGACCGG-3’. The full-length chicken CXCR4 ORF was then amplified by PCR from the cDNA library with the following specific primers located 5’ and 3’ of the start and stop sequences, respectively: sense, 5’-CAGTATCGGCGGAATTCCGGCTCGGAGTAT-3’ and antisense, 5’-AAAGTGTCTATTTATGAAGGGATGGCA-3’. The PCR protocol used to obtain both the 5’ and 3’ ends and the full-length ORF was 1 cycle of 5 min at 94°C, 30 cycles of 30 s at 94°C, 30 s at 65°C, and 1 min at 70°C, and an extension time of 10 min at 72°C. Amplification was performed as described above except that cDNA library DNA was used as template. The PCR product was cloned into pCR2.1, sequenced on both strands, and subcloned between the HindIII and XbaI sites of the mammalian expression plasmid pcDNA3.1. The resulting plasmid was named pcDNA3.chCXCR4.

RNA analysis
Total RNA was isolated from chicken leukocytes with the RNA STAT-60 procedure (Tel-Test, Friendswood, TX). A Northern blot was prepared by standard methods and was prehybridized in Hybrisol I (Oncor) for 20 min at 37°C and hybridized overnight at 37°C with a CXCR4 ORF probe (~1.5 million specific counts/mL), which was labeled with [32P]dCTP using a random primer-labeling kit (Boehringer Mannheim). Blots were washed at high stringency (0.1x SSPE/0.1% SDS for 20 min at 60°C) and exposed for 7 days.

Total RNA was extracted using TRIzol reagent from frozen tissues isolated from 2-week-old chicks and chicken embryonic fibroblasts, untreated or previously treated with stress-inducing agents: thrombin, phorbol dibutyrate, lipopolysaccharide (LPS), and Rous Sarcoma virus (RSV). Reverse transcriptase (RT)-PCR was performed in one tube using the Promega Access RT-PCR System as described in the protocol. Eight hundred and fifty nanograms of template RNA was used in the RT-PCR procedure: first strand synthesis for 45 min at 48°C, then 5 min at 94°C to inactivate the reverse transcriptase, followed by 40 cycles of 45 s at 94°C, 1 min at 60°C, and 90 s at 74°C with a 7-min extension at 74°C. A 460-bp PCR product was produced using two primers: 5’-TCTGTGGCTGACCTCCTCTTTGT-3’and 5’-TCTGGTGGCCTTTTGAATGTGAC-3’. RT-PCR products were analyzed on a 1.2% agarose gel, and the density of the bands was measured by densitometry using the NIH Image Program.

Expression of chicken CXCR4
CHO-K1 cells, maintained in Ham’s F-12 medium (Life Technologies, Gaithersburg, MD) containing 1.5 g/L sodium bicarbonate and 10% fetal bovine serum (FBS), were electroporated with 25 µg of pcDNA3.chCXCR4 plasmid DNA using a GenePulser (Bio-Rad Laboratory, Hercules, CA). Cell colonies resistant to 2 g/L G-418 (GIBCO-BRL) were chosen and expanded in Ham’s F-12 medium with 1.5 g/L sodium bicarbonate, 10% FBS, and 2 g/L G-418. Total RNA was isolated from 30 individual G-418-resistant clones with the RNA STAT-60 method (Tel-Test, Friendswood, TX). Approximately 10 µg of RNA was separated on a denaturing gel, transferred to a nylon membrane, and UV cross-linked. Blots were hybridized with chicken CXCR4 ORF labeled with [32P]dCTP, washed at high stringency (65°C for 20 min in 0.1x SSPE/0.1% SDS), and exposed overnight.

Intracellular [Ca2+] measurements
The cells used in the calcium flux experiments were either parental or transfected CHO-K1 cells grown to confluence in 170-cm2 flasks or freshly isolated chicken blood leukocytes. Cells were washed once with PBS, then incubated with 10 mL of PBS containing 2.5 µM Fura-2/AM (Molecular Probes, Eugene, OR) for 1 h at 37°C in the dark. Cells were harvested and washed twice with Hanks’ balanced salt solution (HBSS). Then 4 x 106 cells in 2 mL HBSS were placed in a continuously stirred cuvette at 37°C in a fluorimeter (Photon Technology, South Brunswick, NJ). Data were recorded every 200 ms as the relative ratio of fluorescence emitted at 510 nm after sequential excitation at 340 and 380 nm. Chemokines used were SDF-1, macrophage inflammatory protein (MIP)-1{alpha}, MIP-1ß, fractalkine, eotaxin, and monocyte chemotactic protein-3 (Peprotech, Rocky Hill, NJ). Where indicated, 250 ng/mL of pertussis toxin was added 3 h before assay.

Chemokine binding assay
Cells were grown to confluency in a 24-well plate in G-418 selection medium. Then they were washed twice in situ in PBS and incubated for 1 h at 4°C in 200 µL total volume of binding buffer [RPMI supplemented with 1% bovine serum albumin (BSA) and 20 mM HEPES] containing 0.1 nM 125I-labeled SDF-1 (New England Nuclear, Boston, MA) with or without unlabeled SDF-1, interleukin (IL)-8, MIP-1{alpha}, or fractalkine. Cells were then washed three times in PBS, lysed with 0.1% SDS, and radioactivity was measured in a Cobra II Gamma counter.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of an avian CXCR4 homolog
Using the human CXCR4 ORF sequence as a probe, Southern blot hybridization of genomic DNA under low stringency conditions revealed a specific band in human and chicken, but not Drosophila, zebrafish (D. rerio), or C. elegans (Fig. 1 ). Using degenerate primers based on the sequences of the mouse and human CXCR4 ORFs corresponding to TMD3 and TMD6, we were able to amplify and clone a 392-bp fragment from chicken genomic DNA. Using specific sequence and vector primers, we obtained the corresponding 5’ and 3’ ends from a chicken cDNA library, and subsequently amplified and cloned a full-length cDNA, named chCXCR4, from the chicken library (GenBank accession no. AF294794).



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  		Figure 1. Phylogenetic analysis of CXCR4. A blot containing EcoRI cut genomic DNA from the indicated species was hybridized with a human CXCR4 ORF probe, washed at low stringency, and exposed overnight. Relevant band sizes are indicated to the left.

 
The longest chCXCR4 ORF contains 359 codons, compared with 352 codons for human CXCR4. The predicted first codon is flanked by a sequence that fits the consensus rules for translation initiation, including an A at the -3 position and a G at the +1 position (AGCATGG). At the nucleotide level, the chCXCR4 ORF is 74 and 71% identical to human and mouse CXCR4, respectively, 71% identical to a frog CXCR4-like sequence deposited in GenBank (accession no. Y17894), and 62 and 64% identical to previously reported fish CXCR4-like sequences from C. carpio and O. mykiss, respectively (Table 1 ). At the amino acid level, chCXCR4 is 82 and 78% identical to human and mouse CXCR4, respectively, 75% identical to the frog homolog, and 60 and 63% identical to the fish sequences from C. carpio and O. mykiss, respectively (Table 1) . Moreover, chCXCR4 has a similar length, seven hydrophobic domains consistent with transmembranous topography, an acidic domain amino-terminal to the first putative transmembrane domain (TMD), a serine-rich domain carboxy-terminal to predicted TMD 7, conserved cysteines, and the highly conserved DRYLAIVHA sequence found at the carboxy terminus of TMD 3 of human, mouse, and frog CXCR4 (Fig. 2 ). The next most highly related human sequence to chCXCR4 is CXCR3 (36% amino acid identity).


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  		Table 1. Structural Relationships of CXCR4-Like Sequences in Vertebrates

 


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  		Figure 2. Structural relationship of vertebrate CXCR4 homologs. Multiple sequence alignment. Identities are shaded at each aligned position. Dashes indicate gaps in the alignment. The length is indicated by the number at the end of each sequence. The sequences were obtained from the following sources: H. sapiens (Swissprot no. P30991), Mus musculus (Swissprot no. P70658), G. gallus (this article and GenBank no. AF294794), X. laevis (GenBank no. Y17885), C. cyprinus ([13 ], GenBank no. AB012310), and O. mykiss ([14 ], GenBank no. AJ001039), and were aligned using the ClustalW 1.74 program (http://molbio.info.nih.gov/molbio/gcglite/clustal17.html).

 
The chicken CXCR4 cDNA contains 59 nucleotides 5’ of codon 1, and 188 nucleotides 3’ of the stop codon. Although the 3’-untranslated sequence is AT-rich, it does not contain a polyadenylation signal or a poly-A tail, suggesting that the cDNA is not full-length. Two partial chicken T cell cDNA sequences identical to ours have been deposited in GenBank. One extends from nucleotide no. 9 of the 5’-UTR through nucleotide no. 340 of the ORF (accession no. AI981253). The second extends from nucleotide no. 1002 of the ORF through nucleotide no. 1325 of the 3’-UTR (accession no. AI980331).

Human SDF-1 is an agonist at chCXCR4
To assess the function of the cloned chicken cDNA, we created a stably transfected CHO-K1 cell line. Of 30 colonies eight expressed chCXCR4 mRNA by Northern hybridization. Colony no. 3 had the highest expression, and was used for functional studies.

In a calcium flux assay, cells expanded from colony no. 3 responded to human SDF-1, the ligand for CXCR4, in a dose-dependent manner (EC50 ~5 nM; saturation at 200 nM; Fig. 3A ). We confirmed the absence of functional CXCR4 in the parental cells by their inability to respond to SDF-1 (Fig. 3B , top tracing). Moreover, the activity was specific for SDF-1 as compared to the lack of responsiveness to MIP-1{alpha}, MIP-1ß, fractalkine, eotaxin, and MCP-3 tested at 100 nM (Fig. 3B , tracing 2). When sequentially stimulated with 200 nM SDF-1, the second stimulation did not produce a response, suggesting complete homologous desensitization of the receptor by the first stimulation (Fig. 3B , tracing 3). The SDF-1-induced activity was completely abolished by pretreatment of the cells with pertussis toxin (Fig. 3B , tracing 4), strongly suggesting that chCXCR4 is coupled to a Gi-type G protein. Thus, by genetic gain of function and pharmacological criteria, we conclude that human SDF-1 is an agonist at chCXCR4 in CHO-K1 cells. Nevertheless, chCXCR4-transfected CHO cells did not respond chemotactically to human SDF-1 (data not shown).



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  		Figure 3. chCXCR4 is a functional receptor for human SDF-1. Calcium mobilization was measured in the indicated cell types activated with the indicated substances at the times marked by arrows. A) Dose response of SDF-1 in chCXCR4 transfected CHO-K1 cells. B) Specificity and mechanism. All tracings correspond to responses of chCXCR4-transfected CHO-K1 cells with the exception of the top tracing, which is for untransfected cells. In the bottom tracing, cells were preincubated in pertussis toxin 250 ng/mL for 3 h. Data are from a single representative experiment repeated at least three times.

 
Consistent with the calcium flux results, human 125I-SDF-1 bound specifically to chCXCR4-transfected CHO-K1 cells (Fig. 4A ). SDF-1 was able to reduce total cell-associated radioactivity in this assay in a dose-dependent manner (IC50 ~10 nM), whereas the chemokines IL-8, MIP-1{alpha}, and fractalkine at 1 µM had little if any effect (Fig. 4B) . Cold SDF-1 up to 50 nM failed to compete for radiolabeled SDF-1 binding to untransfected cells in both of two experiments (Fig. 4A) . At 100 nM cold SDF-1, cell-associated radioactivity was reduced by a similar amount in both untransfected and transfected cells. This is consistent with previously reported results describing a low-affinity, non-signaling SDF-1 binding site on Chinese hamster ovary (CHO) cells involving heparan sulfates [27 ]. This background problem makes SDF-1 binding studies difficult in CHO-K1 cells, however, other cell lines generally used for chemokine receptor expression studies have the even greater problem of high endogenous CXCR4 expression. Despite this problem, our data show a clear difference in specific binding at concentrations of cold SDF-1 below 100 nM between parental and chCXCR4-transfected cells.



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  		Figure 4. Human SDF-1 is a ligand for chCXCR4 expressed in CHO-K1 cells. Competitive binding with 0.1 nM 125I-SDF-1 and the indicated unlabeled chemokines of parental CHO-K1 cells and cells expressing chCXCR4. A) Different concentrations of unlabeled SDF-1{alpha}. B) 1 µM of unlabeled chemokines. Each data point is from a single representative experiment done in duplicate and repeated twice.

 
To test whether SDF-1 is also an agonist at native chCXCR4 we examined chicken peripheral blood leukocytes because human leukocytes express high levels of human CXCR4. When chicken leukocytes were analyzed by Northern hybridization using a chCXCR4 probe, a specific 2-kb RNA band was detected (Fig. 5A ). Consistent with this, human SDF-1 induced a calcium flux response in chicken blood-derived leukoctyes in a dose-dependent manner (EC50 ~10 nM; saturation at 100 nM) from one of three animals tested (Fig. 5B) . These values are close to those reported for SDF-1 activation of human CXCR4 [26 ] and chCXCR4 (Fig. 3) . As with chCXCR4-transfected CHO-K1 cells, when chicken leukocytes were sequentially stimulated with 100 nM SDF-1, the second stimulation did not produce a response, suggesting complete homologous desensitization of the receptor by the first stimulation (Fig. 5C) . However, like chCXCR4-transfected CHO-K1 cells, chicken leukocytes did not respond chemotactically to human SDF-1. The correspondence of these functional studies suggests that the SDF-1 signaling pathway in chicken peripheral blood-derived leukocytes is mediated by chCXCR4.



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  		Figure 5. Chicken blood leukocytes express chCXCR4 and respond to human SDF-1. A) chCXCR4 mRNA expression. A Northern blot with total RNA isolated from chicken peripheral blood-derived leukocytes was hybridized with a chCXCR4 ORF probe, washed at high stringency, and exposed overnight. Band size in kb is indicated to the right. Each lane corresponds to leukocyte RNA from a separate chicken. B) SDF-1 induces calcium flux in chicken leukocytes. Calcium mobilization was measured in chicken blood leukocytes activated with the indicated concentrations of SDF-1 at the times marked by arrows. C) Homologous desensitization of SDF-1-induced calcium flux in chicken blood leukocytes.

 
Analysis of the chCXCR4 gene
Southern blot analysis of chicken genomic DNA digested with a panel of restriction enzymes specific for 6-bp recognition sequences revealed one to two bands in each case (Fig. 6 ). The two PstI fragments can be explained by the presence of a PstI site 841 bp downstream of the start codon. This pattern suggests that in this species, as in human, there is only one copy of the CXCR4 gene and there are no closely related genes.



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  		Figure 6. chCXCR4 is a single copy gene with no close homologues. A blot with G. gallus genomic DNA cut with the enzymes listed at the top of each lane was hybridized with a chCXCR4 ORF probe, washed at high stringency, and exposed overnight. Band sizes in kilobases are indicated to the left.

 
Tissue distribution of chCXCR4 mRNA
Using a sensitive semi-quantitative RT-PCR assay, we detected relatively high levels of chCXCR4 mRNA in brain and bursa, and moderate levels in small and large intestine and liver; mRNA was not detected in stomach or pancreas (Fig. 7A ). This pattern is similar to human CXCR4 distribution. chCXCR4 mRNA was also detected in chicken embryonic fibroblasts, where it could be up-regulated by lipopolysaccharide, but not by thrombin, phorbol dibutyrate, or Rous Sarcoma virus (Fig. 7B) .



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  		Figure 7. Expression and regulation of chCXCR4 in normal tissues and stress-induced fibroblasts. RNA from (A) organs isolated from 2-week-old chicks and (B) unstimulated chicken embryonal fibroblasts (CEFs) or CEFs stimulated for 6 h with phorbol ester (100 nM), thrombin (9 units/mL), lipopolysaccharide (LPS; 10 µg/mL), or from CEFs transformed with Rous sarcoma virus (tCEFs) was measured as described in Materials and Methods. Equal amounts of RNA for the RT-PCR reaction were determined by both spectrophometric analysis and by gel electrophoresis. Graphs below the photos show densitometric analysis of the 460-bp band performed using NIH image software. Relevant band sizes are indicated to the side.

 
Phylogenetic conservation of chCXCR4
To further empower the phylogenetic search for ancestral CXCR4 we next repeated the cross-hybridization analysis carried out previously using a human CXCR4 probe, now using the chCXCR4 ORF probe under low-stringency hybridization and washing conditions. In contrast to our results with the human probe, we now observed cross-hybridizing EcoRI fragments in human and zebrafish (Fig. 8 ). The zebrafish bands are very weak but they are not artifactual because we have cloned a zebrafish CXCR4 homolog [unpublished results]. The chicken and zebrafish sequences are highly divergent, which accounts for the weak cross-hybridization signal seen in Figure 8 . This may be the zebrafish counterpart to a CXCR4-like gene cloned previously from carp [13 ].



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  		Figure 8. chCXCR4 cross-hybridization in other species. A blot containing EcoRI cut genomic DNA from G. gallus, H. sapiens, and D. rerio (zebrafish) was hybridized with a chCXCR4 ORF probe and washed at low stringency. The chicken lane was exposed overnight while the others were exposed for 5 days. Relevant band sizes are indicated to the right.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have cloned and characterized an avian homolog of human CXCR4, the first non-viral, non-mammalian chemokine receptor with demonstrated function. This extends previous reports or database entries of genes and cDNAs from frog, fish, and chicken related to mammalian CXCR4 and other chemokines and chemokine receptors, but not yet studied functionally. In particular, three other chicken chemokine receptor-like sequences have been deposited in the public databases, including CXCR4 and CCR5 (accession nos. AW239706, AF029369, AI979954, AI979976), but most of these are incomplete. The evidence that the molecule we cloned may be the chicken CXCR4 ortholog is its high sequence similarity to human CXCR4 versus other human chemokine receptors and related orphan receptors, and its ability to bind the same ligand as human CXCR4. Thus a functional chemokine system probably exists in birds, and possibly in frog and fish. To date, unlike other components of the innate immune system, there is no evidence that a chemokine system exists in invertebrates [28 29 30 31 32 ]. In particular, we were unable to identify chemokine or chemokine receptor-like sequences by searching the recently published Drosophila genome sequence [33 ].

This study was facilitated by the unusually high sequence conservation of CXCR4 relative to other chemokine receptors, which allows it to be used successfully as a cross-hybridization probe across large species gaps, as well as by the high sequence conservation of its ligand, SDF-1, which allowed retention of agonist activity across large species gaps. We and others have also shown that chicken genomic DNA contains sequences that specifically cross-hybridize to human SDF-1, however, they have not yet been cloned or tested functionally [16 ].

The chemokine signaling system is complex, both with respect to repertoires of ligands and receptors as well as their interactions, which are highly promiscuous [1 ]. Chemokine and chemokine receptors most likely expanded from primordial ancestors by gene replication. The CXCR4 gene may be most closely related to the ancestral receptor. From the analysis shown in Table 1 , however, it is apparent that the sequence of CXCR4 has undergone differential rates of change throughout vertebrate evolution, implying differential selective pressures. Understanding the nature of those pressures will require precise delineation of the biochemical and biological function of CXCR4 in each species. In this regard, mammalian CXCR4, which is ubiquitously expressed in mammalian organs, has been shown to function as a regulator of leukocyte chemotaxis, B cell differentiation, and development of bone marrow, heart, blood vessels, and cerebellum, and HIV replication [16 17 18 19 20 21 22 23 24 ]. Chicken CXCR4 is expressed in the bursa, the site for B cell development, as well as in brain, liver, small and large intestines, chicken embryonic fibroblasts, and chicken blood leukocytes. Expression of a putative fish CXCR4 ortholog has been reported in brain, spleen, liver, gill, head kidney, and in the blood [14 ].

chCXCR4, like its mammalian counterparts, appears to couple to a Gi protein as suggested by pertussis toxin inhibition of signaling. However, we have not been able to demonstrate chemotactic function for chCXCR4 as assessed in human SDF-1-stimulated CHO-K1 cells transfected with chCXCR4 and in chicken blood leukocytes demonstrated to express endogenous chCXCR4 mRNA and to respond by calcium flux to human SDF-1. CHO cells, have also been used in one report for studying chemotactic signaling by the N-formylpeptide receptor, so it is possible that human SDF-1 is sufficiently divergent from chicken that it fails to induce coupling to this pathway [34 ]. Alternatively, CHO cells may simply not be sufficiently motile to be informative in this assay for all receptors, or the expression level of the receptor may be too low to observe this response. Unfortunately, mammalian cell lines typically used for chemotaxis studies already express endogenous CXCR4 and cannot be used to study chCXCR4. chCXCR4 also appears to differ from mammalian CXCR4 in its pattern of gene expression. In particular, chCXCR4 may be up-regulated by inflammatory stimuli such as lipopolysaccharide, at least in embryonal fibroblasts, whereas this effect was not observed in mammalian neuronal cells and macrophages [35 , 36 ]. Results in mammalian fibroblasts have not been reported. Other types of stress-inducing stimuli tested did not affect chCXCR4 expression in chicken fibroblasts.

In conclusion, we have cloned and demonstrated functional activity for a chicken homolog of CXCR4 that is expressed in many chicken organs, as well as in embryonic fibroblasts and blood leukocytes. These results extend the functional chemokine system beyond mammals and into avian species. Although chCXCR4 is clearly closely related to human CXCR4 in structure and biochemical function, additional work will be needed to test whether it shares biological function.

Note added in proof: Moepps et al. have recently shown that Xenopus laevis CXCR4 homologue responds functionally to human SDF-1 [37 ], extending the chemokine system to amphibians.

Received April 22, 2000; revised September 30, 2000; accepted October 3, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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