The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Braun, M.
Right arrow Articles by Moepps, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Braun, M.
Right arrow Articles by Moepps, B.
The Journal of Immunology, 2002, 168: 2340-2347.
Copyright © 2002 by The American Association of Immunologists

Xenopus laevis Stromal Cell-Derived Factor 1: Conservation of Structure and Function During Vertebrate Development1

Mike Braun*, Markus Wunderlin*, Kathrin Spieth*, Walter Knöchel{dagger}, Peter Gierschik2,* and Barbara Moepps*

Departments of * Pharmacology and Toxicology and {dagger} Biochemistry, University of Ulm, Ulm, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transmembrane signaling of the CXC chemokine stromal cell-derived factor-1 (SDF-1) is mediated by CXCR4, a G protein-coupled receptor initially identified in leukocytes and shown to serve as a coreceptor for the entry of HIV into lymphocytes. Characterization of SDF-1- and CXCR4-deficient mice has revealed that SDF-1 and CXCR4 are of vital developmental importance. To study the role of the SDF-1/CXCR4-chemokine/receptor system as a regulator of vertebrate development, we isolated and characterized a cDNA encoding SDF-1 of the lower vertebrate Xenopus laevis (xSDF-1). Recombinant xSDF-1 was produced in insect cells, purified, and functionally characterized. Although xSDF-1 is only 64–66% identical with its mammalian counterparts, it is indistinguishable from human (h)SDF-1{alpha} in terms of activating both X. laevis CXCR4 and hCXCR4. Thus, both xSDF-1 and hSDF-1{alpha} promoted CXCR4-mediated activation of heterotrimeric Gi2 in a cell-free system and induced release of intracellular calcium ions in and chemotaxis of intact lymphoblastic cells. Analysis of the time course of xSDF-1 mRNA expression during Xenopus embryogenesis revealed a tightly coordinated regulation of xSDF-1 and X. laevis CXCR4. xSDF-1 mRNA was specifically detected in the developing CNS, incipient sensory organs, and the embryonic heart. In Xenopus, CXCR4 mRNA appears to be absent from the heart anlage, but present in neural crest cells. This observation suggests that xSDF-1 expressed in the heart anlage may attract cardiac neural crest cells expressing CXCR4 to migrate to the primordial heart to regulate both septation of the cardiac outflow tract and differentiation of the myocardium during early heart development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chemokines constitute a family of low molecular mass (8–11 kDa) and structurally related proteins, with conserved cysteines linked by disulfide bonds (1, 2, 3). They are involved in regulating a wide array of leukocyte functions, including leukocyte chemotaxis, adhesion, and transendothelial migration; they modulate angiogenesis and hematopoiesis; and they block HIV entry into target cells (4, 5, 6, 7, 8). It has become evident that chemokines play fundamental roles in development and homeostasis, and function not only in cells of the immune system, but in many different cell types, including various cells of the CNS (9) or endothelial cells (10). Four subfamilies, C, CC, CXC, and CX3C chemokines, are distinguished based on the relative position of conserved cysteine residues (1, 2).

Stromal cell-derived factor 1 (SDF-1),3 originally identified as a pre-B cell stimulatory factor (11, 12), is a CXC chemokine produced by many cells and tissues (13, 14, 15). The primary structure of SDF-1 is remarkably conserved across species. Thus, human (h) and feline SDF-1 are identical in amino acid sequence (15, 16). This high degree of sequence identity between species has previously been taken to suggest that almost all SDF-1 residues are required for biological activity. Although the genes for other known hCXC chemokines are located on chromosome 4, the gene encoding hSDF-1 is present on chromosome 10 (15). Two isoforms, SDF-1{alpha} (89 aa) and SDF-1{beta} (93 aa), differing only by the presence of four additional amino acids at the very C terminus of the longer isoform are generated from a single gene by differential RNA splicing (14, 15). Very recently, a third isoform of SDF-1, designated SDF-1{gamma} (119 aa), has been identified in the rat (17). SDF-1{gamma} is identical to rat SDF-1{alpha} in its first 89 residues, but is 30 or 26 residues longer than rat SDF-1{alpha} and {beta}, respectively. The functional significance of the existence of various mammalian SDF-1 isoforms is currently unknown. SDF-1 is known to play a critical role in the regulation of trafficking and transendothelial migration of leukocytes and in the control of proliferation and differentiation of several cell types, including hematopoietic and neural cells (7, 18). Transmembrane signaling of SDF-1 is mediated by CXCR4 (19, 20), a heterotrimeric G protein-coupled chemokine receptor initially identified in leukocytes, and known to serve as a coreceptor for the entry of T cell-tropic and dual-tropic HIV into CD4+ lymphocytes (8). Although other CXC chemokines can compete for binding to and activation of several distinct chemokine receptors, SDF-1 is unusual in that it activates a single receptor, CXCR4. Inactivation of the genes encoding SDF-1 or CXCR4 in mice caused defects of B lymphopoiesis, myelopoiesis, gastrointestinal vascularization, and heart ventricular septum formation in the developing embryo (21, 22, 23, 24). These observations suggested that the SDF-1/CXCR4 chemokine/receptor system is of vital developmental importance.

To study the role of chemokines and chemokine receptors as regulators of early vertebrate development, we set out to isolate and characterize their cDNAs from Xenopus laevis, an organism widely used to study vertebrate embryogenesis. Recently, we isolated and characterized a cDNA encoding CXCR4 of X. laevis (xCXCR4; Ref. 25). xCXCR4 mRNA expression was up-regulated during early neurula stages and remained high during early organogenesis. Whole-mount in situ hybridization analysis showed abundant expression of xCXCR4 mRNA in the nervous system, including forebrain, hindbrain, and sensory organs, and in neural crest cells. To identify the ligand which activates xCXCR4 in Xenopus, we have now isolated a cDNA encoding Xenopus SDF-1 (xSDF-1). The encoded polypeptide, designated xSDF-1, was functionally characterized, and the expression of its mRNA was determined during embryonic development and in the adult frog.

The results revealed that xSDF-1 differs considerably in primary structure from its mammalian counterparts, but is nevertheless undistinguishable from hSDF-1 in terms of activating both hCXCR4 and xCXCR4. Our findings not only allow identification of key residues of SDF-1 and CXCR4 involved in agonist binding and receptor activation, but also show that these residues have been maintained over a period of at least 325 million years, which is the approximate evolutionary distance between Xenopus and living mammals (26). The latter is in support of a pivotal role of this chemokine receptor pair in vertebrate development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

The production of baculoviruses encoding xCXCR4, hCXCR4, rat G protein {alpha}i2 subunit, and both human G protein {beta}1 subunit and bovine G protein {gamma}3 subunit is described in Ref. 25 . The N33A analog of hSDF-1{alpha} (27) was prepared by chemical synthesis, and was generously provided by Dr. M. A. Siani (Gryphon Sciences, South San Francisco, CA). [35S]Guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP[S]) was obtained from NEN (Boston, MA). X. laevis embryos were prepared and staged according to Refs. 28 and 29 . A X. laevis spleen cDNA library was obtained from the Resource Center/Primary Database within the German Human Genome Project at the Max-Planck Institute for Molecular Genetics (Berlin, Germany; http://www.rzpd.de).

Screening of a X. laevis cDNA library

The X. laevis spleen cDNA library was probed with a 32P-labeled mouse SDF-1{alpha} cDNA fragment (nt 145–351, European Molecular Biology Laboratory (EMBL)/GenBank Data Libraries accession no. L12029). Positive clones were analyzed by restriction endonuclease mapping and DNA sequencing.

Production of recombinant baculoviruses

The xSDF-1 cDNA was digested with HindIII and EcoRI. The resulting fragment was filled in using Klenow enzyme and subcloned into the SmaI site of the baculovirus transfer vector pVL1393 (Invitrogen, San Diego, CA). The correct orientation of the insert was verified by DNA sequencing. Recombinant baculoviruses were obtained by transfecting Spodoptera frungiperda cells (Sf9 cells; Invitrogen) with a 25/1 mixture of the transfer vector and a modified baculovirus DNA (BaculoGold, BD PharMingen, San Diego, CA), which contains a lethal deletion and is rescued by the DNA of the transfer vector. High-titer stocks of the baculoviruses were obtained by two cycles of amplification in Sf9 cells.

Production of recombinant xSDF-1

For production of recombinant xSDF-1, Trichoplusia ni 5B1-4 cells (High five cells; Invitrogen) were grown at 27°C in suspension culture in Insect-XPRESS medium (BioWhittaker, Walkersville, MD) supplemented with 0.2% (v/v) Pluronic F-68 (Life Technologies, Grand Island, NY), 0.5 mg/ml gentamicin (Life Technologies), and 2.5 µg/ml amphotericin B (Fungizone; Life Technologies) in 1800 ml Fernbach culture flasks. Cells (8 x 108/flask) were incubated for 48 h with recombinant baculovirus in 400 ml medium/flask at 80 rpm on a rotary shaker with an amplitude of 25 mm. The medium containing recombinant xSDF-1 was collected from the cell suspension by centrifugation at 80,000 x g for 30 min at 4°C. The supernatant was passed through 0.22-µm pore size nitrocellulose filters, snap-frozen in liquid nitrogen, and stored at -80°C.

Purification of recombinant xSDF-1

Recombinant xSDF-1 was purified from the culture supernatant by sequential chromatography on Heparin Sepharose High Performance and SOURCE 15S using a Pharmacia ÄKTAexplorer chromatography system (Amersham Pharmacia Biotech, Piscataway, NJ). The filtered supernatant (150 ml, 230 mg of protein) was applied to a 5 ml HiTrap Heparin column (Amersham Pharmacia Biotech) that had been equilibrated with buffer A (10 mM Na2HPO4/NaH2PO4, pH 7.3). The flow rate was 3.5 ml/min. After application of the sample, the column was washed with 60 ml of buffer A and eluted with a linear gradient (50 ml) of NaCl (0–2 M) in buffer A. Fractions of 2 ml were collected and analyzed by SDS-PAGE and CXCR4-mediated [35S]GTP[S]-binding. The active material, which eluted at ~1.1–1.5 M NaCl, was pooled (12 ml, 3.6 mg of protein) and diluted with 10 mM Na2HPO4/NaH2PO4, pH 7.3, to obtain a final NaCl concentration of 0.1 M. The sample was then applied to a 1 ml RESOURCE S column (Amersham Pharmacia Biotech), which had been equilibrated with buffer A. The flow rate was 2 ml/min. After application of the sample, the column was washed with 15 ml of buffer A and eluted with a linear gradient (40 ml) of NaCl (0–1 M) in buffer A. Fractions of 1 ml were collected and analyzed by SDS-PAGE and CXCR4-mediated [35S]GTP[S]-binding. The active material eluted at ~0.5–0.6 M NaCl.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-MS)

Molecular mass determination of proteins was performed by delayed extraction MALDI-MS on a Bruker REFLEX III time-of-flight spectrometer (Bruker-Daltonics, Bremen, Germany) equipped with a UV nitrogen laser (337 nm). The samples were concentrated and desalted using ZipTip C18 pipette tips (0.6 µ l resin) as recommended by the manufacturer (Millipore, Bedford, MA). The samples were eluted from the C18 matrix in acetonitrile/0.1% (v/v) trifluoroacetic acid, 2/1 (v/v). One microliter of this solution was mixed with 1 µl of saturated {alpha}-cyano-4-hydroxycinnamic acid solution in acetonitrile/0.1% (v/v) trifluoroacetic acid, 2/1 (v/v). Spectra were recorded after evaporation of the solvent. The singly and doubly charged ion signals from bovine ubiquitin (Mr = 8565.8510) were used for external mass calibration of all mass spectra.

Membrane preparation of baculovirus-infected insect cells

Sf9 cells were grown at 27°C in 59 cm2 cell-culture dishes in TNM-FH medium (T 1032; Sigma Aldrich, St. Louis, MO) supplemented with 10% FCS and 0.5 mg/ml gentamicin. For production of recombinant receptors and heterotrimeric Gi2, cells were grown to a density of ~60%, and incubated for 1 h at 27°C in 2 ml per dish of medium containing the recombinant baculovirus(es). The cells were then supplemented with 9 ml per dish of fresh medium and maintained in this medium for 48 h at 27°C. Infected cells were suspended in medium, pelleted by centrifugation, and resuspended in 600 µl per dish of ice-cold lysis buffer containing 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 3 µM GDP, 2 µg/ml soybean trypsin inhibitor, 1 µM pepstatin, 1 µM leupeptin, 100 µM PMSF, and 1 µg/ml aprotinin. Cells were homogenized by forcing the suspension six times through a 0.5 x 23 mm needle attached to a disposable syringe. After 30 min on ice, the lysate was centrifuged at 2,450 x g for 45 s to remove unbroken cells and nuclei. A crude membrane fraction was isolated from the resulting supernatant by centrifugation at 26,000 x g for 30 min at 4°C. The pellet was rinsed with 300 µl of lysis buffer, resuspended in 300 µl of fresh lysis buffer, snap-frozen in liquid nitrogen, and stored at -80°C.

[35S]GTP[S] binding

Binding of [35S]GTP[S] to membranes of baculovirus-infected insect cells was assayed as described (30). In brief, membranes (10 µg of protein per sample) were incubated for 60 min at 30°C in a mixture (100 µl) containing 62.5 mM triethanolamine/HCl, pH 7.4, 1.25 mM EDTA, 6.25 mM MgCl2, 95 mM NaCl, 3.75 µM GDP, and 0.34 nM [35S]GTP[S] (1,250 Ci/mmol). The incubation was terminated by rapid filtration through 0.45-µm pore size nitrocellulose membranes (Advanced Microdevices, Ambala Cantonment, India). The membranes were washed and dried, and the retained radioactivity was determined by liquid-scintillation counting. Nonspecific binding was defined as the binding not competed for by 60 µM unlabeled GTP[S].

Measurement of cytosolic free Ca2+ concentration

CCRF-CEM cells (2 x 106 cells) were loaded with fura 2 by incubation for 30 min at 37°C in 1 ml of Ca2+ flux buffer (20 mM HEPES/NaOH, pH 7.4, 1 mM CaCl2, 4.6 mM KCl, and 136 mM NaCl) containing 1 nM of the acetoxymethyl ester of fura 2 (Molecular Probes, Eugene, OR). The cells were washed and resuspended in the same buffer (1 ml), and 5 µl of xSDF-1 in 10 mM Na2HPO4/NaH2PO4, pH 7.3 was added. Fura 2 fluorescence was measured on a fluorescence spectrophotometer (LS50B, PerkinElmer, Wellesley, MA) thermostated at 37°C. Excitation and emission wavelengths were 340/380 and 510 nm, respectively.

Chemotaxis assay

The migration of human acute lymphoblastic leukemia cells (CCRF-CEM, American Type Culture Collection (Manassas, VA) certified cell line 119) was assessed in disposable Transwell trays (Costar, Cambridge, MA) with 6.5-mm diameter chambers and 3-µm pore-size polycarbonate membranes. In brief, chemokines were diluted in HEPES-buffered RPMI 1640 (Life Technologies) supplemented with 10 mg/ml BSA and added to the lower compartments, and CCRF-CEM cells (106 cells) were added in the same medium (100 µl) to the upper compartments. After an incubation for 2 h at 37°C in a humidified CO2 atmosphere (10%), the filters were removed from the chamber, washed in PBS, fixed, and stained with Diff-Quik (Dade Behring, Marburg, Germany) according to the manufacturer’s instructions. The number of cells migrated through the membrane was determined by microscopical examination in three randomly selected fields of three independent Transwell chambers at x40 magnification.

RT-PCR analysis of xSDF-1 mRNA expression

Total RNA was prepared from X. laevis embryos and adult tissues using TRIzol Reagent as recommended by the supplier (Life Technologies). Oligo(dT)-primed sscDNA was made from total RNA after DNase I digestion (amplification grade; Life Technologies) using the SuperScript preamplification system (Life Technologies). The amounts of sscDNA used as templates for the amplification of the xSDF-1 cDNAs were adjusted to similar levels according to the amount of single-stranded X. laevis histone H4 cDNA present in the sample, as determined by semiquantitative PCR. The xSDF-1 cDNA was amplified from these samples by PCR (28 cycles: 94°C for 30 s, 59°C for 45 s, 72°C for 45 s, followed by a single incubation at 72°C for 10 min) using primers P1, 5'-CACAGCTCCAGCCACAACATG-3' (nt 14–34, sense), and P2, 5'-GCCAGAACACTAACAAAGAAATTA-3' (nt 315–338, antisense). The numbering of all oligonucleotides used as primers in this study refers to the nucleotide sequence of xSDF-1 cDNA deposited in the EMBL/GenBank Data Libraries under accession no. AJ278857. The PCR products contained in 8 µl of the reaction volume were fractionated by agarose (1% (w/v)) gel electrophoresis, transferred to a nylon membrane (Biodyne B; Pall, Dreieich, Germany), and sequentially hybridized with radiolabeled xSDF-1 (nt 1–386) and Xenopus histone H4 cDNA probes (nt 1491–1679, EMBL/GenBank accession no. X03017).

Whole-mount in situ hybridization of X. laevis embryos

The localization of xSDF-1 transcripts in Xenopus embryos was analyzed by using the whole-mount in situ hybridization technique (31) with some modifications (28). In brief, embryos were fixed for 90 min at room temperature in freshly prepared MEMPFA(0.1 M MOPS, pH 7.4, 2 mM EGTA, 1 mM MgSO4, and 4% (w/v) paraformaldehyde), and then stored at -20°C in ethanol. Whole-mount in situ hybridization was performed with digoxigenin-labeled antisense cRNA transcribed in vitro using T7 RNA polymerase from a xSDF-1 cDNA fragment (nt 1–341) using the digoxigenin RNA Labeling kit (SP6/T7; Boehringer Mannheim, Indianapolis, IN). Labeled antisense RNA transcripts were localized by alkaline phosphatase-conjugated anti-digoxigenin Abs and Boehringer Mannheim purple substrate (Boehringer Mannheim). After final fixation of the embryos in MEMPFA for at least 2 h, they were bleached in 30% H2O2/methanol (33/67, by volume; 10% H2O2 final), dehydrated in pure methanol, and stored at 4°C in pure methanol. To enhance the transparency of the embryos, they were incubated for 60 min at 20°C in benzylbenzoate/benzyl alcohol (2/1, by volume).

Miscellaneous

Radiolabeled cDNA probes were prepared by priming with random hexanucleotides (32). Blots were hybridized with radiolabeled probes as descibed in Ref. 33 , except that the final wash was done at 64°C in a solution of 2x SSC and 0.1% (w/v) SDS. DNA was sequenced on an ABI Prism 310 Genetic Analyzer using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA Polymerase, FS (Applied Biosystems, Foster City, CA). Protein concentrations were determined according to Bradford (34) using bovine IgG as standard.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of a SDF-1 cDNA from a X. laevis cDNA library

To determine the primary structure of the X. laevis homolog of mammalian SDF-1, a X. laevis spleen cDNA library was screened at low stringency using a radiolabeled cDNA fragment of mouse SDF-1{alpha} as a probe. DNA sequence analysis and comparison of the predicted amino acid sequences of three independent clones to the sequences of known chemokines revealed that all three cDNAs encoded a protein showing high sequence similarity to mammalian SDF-1. One of these cDNAs (Resource Center/Primary Database clone ID: HACHp412I1133Q2) was sequenced by primer walking. The cDNA encoded a polypeptide of 94 aa with a calculated Mr of 10,701, which was tentatively designated as xSDF-1. The 5' noncoding region of this cDNA started at position -31 from the ATG initiation codon. No other in-frame ATG codon was present in the 5' noncoding region. The 3' noncoding region consisted of ~1.9 kb. Fig. 1Go shows an alignment of the amino acid sequences of xSDF-1 and SDF-1{beta} of man (hSDF-1{beta}), mouse (mSDF-1{beta}), and cat (cSDF-1{beta}) as well as KC, the mouse equivalent of human growth-related oncogene (GRO)-{alpha}, GRO-{beta}, and/or GRO-{gamma} (19, 35). KC was the closest relative of xSDF outside the SDF-1 family identified in the National Center for Biotechnology Information databases using the basic local alignment search tool algorithm. xSDF shares 64–66 and 26% identical amino acids with the mammalian SDF-1 polypeptides and KC, respectively. Thus, the degrees of amino acid identity between xSDF-1 and its mammalian counterparts (64–66%) are strikingly lower than the degrees obtained when mammalian SDF-1 polypeptides are compared with each other (92–96%). Analysis of the xSDF-1 sequence using a signal-sequence-detecting algorithm (36) led to the prediction of three potential signal sequence cleavage sites with similar scores between residues Gly21 and Lys22, Cys17 and Leu18, Thr19 and Glu20 (scores: 8.57, 6.71, and 6.57, respectively). Assuming that the former site is the major cleavage site in Xenopus, as it is in the mouse (12), the mature xSDF-1 polypeptide consists of 73 aa with a calculated Mr of 8,468. Although the mature hSDF-1{beta} and cSDF-1{beta} proteins are identical and differ from the mature mSDF-1{beta} by only two residues, mature xSDF-1 differs by 21 residues from its mammalian counterparts (white in Fig. 1Go). Eighteen of the remaining 52 identical residues are also found in the non-SDF-1 CXC chemokine KC (pink in Fig. 1Go), 34 residues are uniquely present in SDF-1 (red in Fig. 1Go).



View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 1. Comparison of the amino acid sequences of X. laevis and mammalian SDF-1. The amino acid sequences of xSDF-1, hSDF-1{beta}, mSDF-1{beta}, cSDF-1{beta}, and mouse KC were aligned using the program CLUSTAL W (53 ) contained in the OMIGA 2.0 software package (Oxford Molecular, Oxford, U. K.). Residues present in all SDF-1 polypeptides, but absent from KC, are shown in red, those present in both SDF-1 and KC are shown in gray. The amino acid sequence numbering starts with the lysine residue present at the amino terminus of mature SDF-1 (12 ). The positions of the structural elements of hSDF-1 (41 ) are indicated. The nucleotide sequence of xSDF-1 has been deposited with the EMBL/GenBank Data Libraries under accession no. AJ278857. The amino acid sequences of hSDF-1{beta}, mSDF-1{beta}, cSDF-1{beta}, and KC correspond to SWISSPROT databank entries P48061, P40224, O62657, and P12850, respectively.

 
Production of the recombinant xSDF-1

To obtain mature xSDF-1 as a recombinant protein, the cDNA was cloned into the baculovirus transfer vector pVL1393 and a recombinant baculovirus was produced. Insect cells were infected with the virus, and recombinant mature xSDF-1 was purified from the culture medium by sequential heparin affinity and cation exchange chromatography. Analysis of the fractions eluting from the cation exchange resin by SDS-PAGE revealed that a mostly homogeneous protein of the expected molecular mass was obtained at ~0.5–0.6 M NaCl (Fig. 2GoA). The purified protein was subjected to molecular mass determination by MALDI-MS (Fig. 2GoB). The measured molecular mass was 8,469.03 Da, which is in excellent agreement with the calculated mass ([M + H]+) of mature xSDF-1 of 73 aa (K1-T73, cf Fig. 1Go) containing four half-cystines (8,469.08 Da). In addition, two peptides with smaller measured molecular masses (8,243.79 and 8,057.52) were present in the same peak. These peptides most likely correspond to the V3-T73 and L5-T73 forms of xSDF-1. The yield of purified protein was typically ~1 µg/ml of starting culture supernatant.



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 2. Purification on RESOURCE S and mass spectrometric analysis of recombinant xSDF-1. A, Recombinant xSDF-1 was partially purified from the culture supernatant of High five insect cells infected with baculovirus encoding xSDF-1 by sequential chromatography on Heparin Sepharose High Performance and SOURCE 15S. Fractions containing xSDF-1 were pooled and subjected to chromatography on RESOURCE S. Aliquots of the indicated fractions were analyzed by SDS-PAGE. Proteins were stained with Coomassie blue (inset). The positions of the molecular mass standards (bovine {alpha}-lactalbumine, 14.4 kDa; bovine aprotinin, 6.5 kDa) are indicated. B, Fraction 24 was analyzed by MALDI-MS. An expansion of the [M + H]+ molecular ion region of the spectrum is shown. The corresponding peptides are indicated. Assuming that the relative amounts of the three nearly identical components can be deduced by comparing the areas under the curve of the corresponding peaks (54 ), we estimate that the L5-T73, V3-T73, and K1-T73 forms of xSDF-1 are present in this preparation at 4, 67, and 29% (w/w), respectively.

 
Functional reconstitution of xCXCR4 and hCXCR4 with heterotrimeric Gi2

To investigate whether xSDF-1 does in fact represent an agonist of CXCR4, including xCXCR4, rCXCR4 of Xenopus (xCXCR4) and man (hCXCR4) were reconstituted with recombinant heterotrimeric Gi2 by coinfection of Sf9 insect cells with baculovirus encoding either xCXCR4 or hCXCR4, and a combination of baculoviruses encoding the Gi2 subunits {alpha}i2 and {beta}1{gamma}3. Receptor-G protein interaction was assayed by measuring the effect of hSDF-1{alpha} and xSDF-1 on the binding of [35S]GTP[S] to insect cell membranes. Fig. 3Go shows that xSDF-1 (200 nM) and hSDF-1{alpha} (200 nM) caused similar (~4-fold) activation of xCXCR4 and hCXCR4. Both xSDF-1 and hSDF-1{alpha} failed to increase [35S]GTP[S]-binding in membranes expressing {alpha}i2·{beta}1{gamma}3 in the absence of xCXCR4 or hCXCR4. No activation of [35S]GTP[S]-binding was observed upon testing xSDF-1 at the same concentration (200 nM) on membranes of insect cells coexpressing other CXCRs, e.g., CXCR1 and 2, and {alpha}i2·{beta}1{gamma}3 (data not shown). Taken together, these results demonstrate that xSDF-1 specifically activates not only Xenopus, but also hCXCR4 and thus, confirm that xSDF-1 is the amphibian counterpart of mammalian SDF-1. Furthermore, the results introduce xSDF-1 as the first functional nonmammalian chemokine to be described.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 3. Activation of xCXCR4 and hCXCR4 by hSDF-1{alpha} and xSDF-1. Sf9 cells were coinfected with baculoviruses encoding either xCXCR4 or hCXCR4, and the G protein subunits {alpha}i2 and {beta}1{gamma}3. Cells were homogenized and fractionated into soluble and particulate fractions, and aliquots of the particulate fractions (10 µg protein/sample) were incubated with 0.34 nM [35S]GTP[S] in the absence or presence of 200 nM SDF-1{alpha} or xSDF-1. The samples were analyzed for bound [35S]GTP[S] by rapid filtration and scintillation counting. Each value represents the mean ± SD of triplicate determinations.

 
Release of intracellular Ca2+ and chemotaxis

There is evidence to suggest that chemically distinct agonists may produce arrays of active states of a single G protein-coupled receptor to differentially activate the G proteins of a given cell, and thus, produce distinct cellular responses (37, 38, 39, 40). Therefore, the effects of xSDF-1 and hSDF-1{alpha} on hCXCR4-mediated responses of intact human cells were examined and compared. Fig. 4Go shows that xSDF-1 induced a rapid and transient rise in the concentration of cytoplasmic free calcium, intracellular Ca2+ concentration, in human acute lymphoblastic leukemia (CCRF-CEM) cells. The intracellular Ca2+ concentration increase was very similar both in terms of its kinetics and its magnitude to the increase observed upon testing hSDF-1{alpha} at the same concentration. xSDF-1 and hSDF-1{alpha} also caused very similar concentration-dependent chemotactic responses of CCRF-CEM cells with maximal effects at ~10 nM SDF-1 (Fig. 5Go). Thus, xSDF-1 and hSDF-1{alpha} are indistinguishable in regulating complex functions of intact cells by activating a single species of CXCR4.



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 4. Effects of xSDF-1 and hSDF-1{alpha} on the concentration of cytosolic free Ca2+ in intact CCRF-CEM cells. Human acute lymphoblastic leukemia (CCRF-CEM) cells were loaded with fura 2. At the times indicated by arrows, xSDF-1 (20 nM) or hSDF-1{alpha} (20 nM) were added. The fluorescence due to intracellular Ca2+ was recorded.

 


View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 5. Stimulation of CCRF-CEM cell chemotaxis by xSDF-1 and hSDF-1{alpha}. The effect of xSDF-1 and hSDF-1{alpha} on chemotaxis of human acute lymphoblastic leukemia (CCRF-CEM) cells was assessed in disposable Transwell trays with 6.5-mm diameter chambers equipped with 3-µm pore size polycarbonate membranes. The chemotactic response to xSDF-1 is plotted as a function of the concentrations of the full-length (K1-T73), active constituent of recombinant xSDF-1 and of full-length (K1-K68) recombinant hSDF-1{alpha}. The values shown represent the mean ± SD of the cell numbers determined in three independent Transwell chambers.

 
Expression of xSDF-1 mRNA in adult X. laevis tissues and in X. laevis embryos

The expression of xSDF-1 mRNA in tissues of adult X. laevis was assessed by RT-PCR analysis (Fig. 6Go). Using this methodology, high levels of xSDF-1 mRNA were detected in spleen, kidney, lung, stomach, testis, and skeletal muscle. Lower levels were observed in liver and heart. Few transcripts were found present in brain and skin. No xSDF-1 mRNA was detected in the ovary. The temporal and spatial pattern of xSDF-1 mRNA expression during embryogenesis was assessed by RT-PCR analysis and whole-mount in situ hybridization (Figs. 7Go and 8Go). RT-PCR analysis of RNA prepared from X. laevis embryos at different stages of embryonic development revealed a sharp rise of xCXCR4 mRNA expression between stages 14 and 18 to a level increasing further until stage 45 (Fig. 7Go). Results obtained by whole-mount in situ hybridization analysis were consistent with this pattern. As shown in Fig. 8GoA, xSDF-1 mRNA expression was up-regulated during organogenesis (stages 21–23) in the anterior part of the embryo, where the regional segregation of the neural tube into fore-, mid-, and hindbrain takes place (29). In stage 32–34 embryos, xSDF-1 mRNA was detected in the mid- and hindbrain, otic vesicles and eyes, and the dorsal fin (Fig. 8GoB). Furthermore, xSDF-1 transcripts were present within the posterior heart anlage, where the pericardial mesoderm begins to form the dorsal mesocardium at stage 32 (29). At stages 39–40, xSDF-1 mRNA was found highly expressed in the proctodeum located in the posterior part of the embryo (Fig. 8GoA).



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 6. RT-PCR analysis of xSDF-1 mRNA expression in tissues of adult X. laevis. Total RNA was prepared from adult X. laevis spleen, kidney, lung, liver, heart, brain, stomach, testis, ovary, muscle, and skin and used as template for RT-PCR. The amounts of sscDNA used as templates for the amplification of the xSDF-1 cDNA (upper panel) were adjusted to similar levels according to the amount of single-stranded X. laevis histone (H4) cDNA present in the sample (lower panel), as determined by semiquantitative PCR. The amplified DNA fragments were fractionated by agarose (1% (w/v)) gel electrophoresis, transferred to a nylon membrane, and hybridized with radiolabeled xSDF-1 (upper panel) or X. laevis histone H4 (lower panel) cDNA fragments. A sample containing no single-stranded template cDNA was analyzed for comparison (Control).

 


View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 7. RT-PCR analysis of xSDF-1 mRNA expression in X. laevis embryos. Total RNA was prepared from from X. laevis embryos (stages 2–34; Ref. 29 ), and used as template for RT-PCR. The amounts of sscDNA used as templates for the amplification of the xSDF-1 cDNA (upper panel) were adjusted to similar levels according to the amount of single-stranded X. laevis histone (H4) cDNA present in the sample (lower panel), as determined by semiquantitative PCR. The amplified DNA fragments were fractionated by agarose (1% (w/v)) gel electrophoresis, transferred to a nylon membrane, and hybridized with radiolabeled xSDF-1 (upper panel) or X. laevis histone H4 (lower panel) cDNA fragments. A control sample containing no single-stranded template cDNA was analyzed for comparison (C).

 


View larger version (88K):
[in this window]
[in a new window]
  		FIGURE 8. Whole-embryo in situ analysis of xSDF-1 mRNA expression during X. laevis embryogenesis. A, Digoxigenin-labeled xSDF-1 antisense RNA was prepared and hybridized to Xenopus embryos (stages 21–23, 32–34, and 39–40). For enhancement of transparency, the embryos were treated with benzylbenzoate/benzyl alcohol. B, Stage 32–34 embryo (e, eye; b, brain; ov, otic vesicle; and h, heart anlage).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have isolated and characterized a cDNA encoding a X. laevis homolog of mammalian SDF-1. The encoded protein consists of 94 aa and is only 64–66% identical to its mammalian counterparts. The degree of amino acid identity is particularly low within the predicted signal sequence, Met-20-Gly-1, where only 4 of 21 residues (excluding the initiating methionine) are shared between Xenopus and mammalian SDF-1. The three-dimensional structure of hSDF-1 is made up of three antiparallel {beta}-strands ({beta}1, residues 24–31; {beta}2, residues 35–42; {beta}3, residues 45–49; cf Fig. 1Go) followed by a C-terminal {alpha}-helix (residues 56–64) that is packed against the {beta}-sheet (41, 42). The first {beta}-sheet is preceeded by a disordered amino-terminal region (residues 1–8), an extended loop (residues 12–18), and a single turn of a 310 helix (residues 19–22; Refs. 41 and 42). An alignment of the primary structures of Xenopus and mammalian SDF-1 with these structural elements revealed that the degree of sequence identity between the mature SDF-1 polypeptides is particularly high for the C-terminal {alpha}-helix (100%), followed by the amino terminus, {beta}1, and {beta}2 (each 88%), the extended loop (57%), {beta}3 (40%), and the 310 helix (25%; Fig. 1Go).

The high degree of sequence identity within the region corresponding to the flexible amino-terminus of hSDF-1 is consistent with the observation that this region is directly involved in high-affinity binding of SDF-1 to and activation of hCXCR4 (12, 42). The fact that a hydrophobic valine residue is present in position 6 of xSDF-1 in place of the polar serine present in mammalian SDF-1 implies that this residue is not involved with the agonist-receptor interaction. Crump et al. (42) have recently proposed a two-site model for the interaction of SDF-1 with mammalian CXCR4 (43). According to this model, binding of SDF-1 is initiated by a direct interaction ("docking step") between a sequence motif immediately adjacent to the second cysteine residue, R12FFESH (site 1), with the amino-terminal segment of CXCR4. The docking permits access of the flexible amino terminus of SDF-1 (site 2) to a more buried receptor site. The second binding step induces a conformational change of the receptor transmembrane helices that allows intracellular G protein activation. Very recently, the major conformation of the amino-terminal 17 residues of mature mammalian SDF-1 (K1PVSLSYRCPCRFFESH) was determined by nuclear magnetic resonance, and found to consist of two similar {beta}-turns of the {beta}-{alpha}R type made up of residues 5–8 and 11–14 (44). The authors noticed that the CRF portion of the second {beta}-turn is a partial palindrome of the tail of the 9-mer sequence (K1PVSLSYRC) involved in the formation of the first {beta}-turn. This might explain the enhanced affinity of a 9-mer dimer (residues 1–9 linked by a disulfide bond at residue 9) relative to monomeric 9-mer to activate the receptor (45). The fact that this palindome is perfect in xSDF-1 is in support of this notion. In any case, two of six residues of the "RFFESH motif," corresponding to Phe13 and His17 of mammalian SDF-1, appear to be dispensable for the docking process, since these residues are replaced by tyrosine and asparagine, respectively, in xSDF-1. In this context, the RFFESH motif might be more appropriately referred to as R(Y/F)FES motif.

The high degree of sequence identity between Xenopus and mammalian SDF-1 in regions downstream of the R(Y/F)FES motif is surprising in light of previous observations demonstrating that the first 17 residues of hSDF-1 are sufficient for binding to and activation of CXCR4 (42, 45, 46). Compared with the low degree of sequence conservation within the presumed signal peptide, the conservation is particularly striking in the regions corresponding to the {beta}1 and {beta}2 strands and to the C-terminal {alpha}-helix, all of which contain a high proportion of the residues present in both Xenopus and mammalian SDF-1, but absent from the closest SDF-1 relative, KC (red in Fig. 1Go). The high degree of conservative evolutionary pressure to maintain these residues over the past 325 million years, which is the approximate evolutionary distance between Xenopus and living mammals (26), strongly suggests that these residues make up functionally important parts of the SDF-1 molecule. Although it seems possible that some of the conserved residues are involved in maintaining the overall tertiary structure, and possibly the stability of SDF-1, it is important to note that a chimeric CXC chemokine, designated GROH2, sharing with SDF-1 only 6 of the 24 residues specifically present in SDF-1 (red in Fig. 1Go) downstream of the R(Y/F)FES motif (His25, Ile28, Thr31, Asn33, Glu63, Asn67) bound to and activated CXCR4 with Kd and EC30 values corresponding closely to the values of native SDF-1{alpha} and SDF-1{beta} (42). Thus, the conserved residues may be involved in serving functions of SDF-1 other than interaction with CXCR4 and maintenance of protein structure and/or stability. For example, structure function analysis of human SDF-1{alpha} has shown that three {beta}1-strand residues of hSDF-1{alpha}, Lys24, His25, and Lys27 either form or are essential elements of a heparan sulfate binding site, which is distinct from the site required for binding to, and signaling through hCXCR4 (47). Very recently, three additional residues, Lys1, Arg41, and Lys43, have been shown to also participate in glycosaminoglycan binding of SDF-1{alpha} (48). All six residues are conserved between Xenopus and mammalian SDF-1 (cf. Fig. 1Go). Therefore, it seems possible that the conserved residues downstream of the R(Y/F)FES motif are involved in mediating the interaction with glycosaminoglycans.

Analysis of xSDF-1 mRNA expression in tissues of adult X. laevis revealed high transcript levels in spleen, kidney, lung, liver, heart, stomach, testis and skeletal muscle. Excepting lung and testis, this expression pattern corresponds closely to the pattern observed for tissues of adult mouse and man (13, 15). Consistent with the CXCR4 mRNA expression pattern in adult Xenopus (25), xSDF-1 mRNA is particularly abundant in the spleen, which appears to be the principal site of B cell differentiation in adult Xenopus (49). This finding is in support of the notion that the xSDF-1/xCXCR4 chemokine/receptor pair regulates adult amphibian B cell differentiation (25).

Analysis of the time course of xSDF-1 mRNA expression during Xenopus embryogenesis revealed the appearance of xSDF-1 mRNA between stages 14 and 18, i.e., during neurulation (29). This pattern corresponds very closely to the time course described for the receptor xCXCR4 (25), suggesting that expression of the ligand xSDF-1 and of its receptor xCXCR4 is tightly coordinated during Xenopus embryogenesis. Although the spatial distribution of xSDF-1 mRNA appears to be less discrete than the distribution of xCXCR4 mRNA (25), xSDF-1 mRNA was specifically detected in several organ systems, including the developing CNS, incipient sensory organs, and the embryonic heart. This distribution is consistent with the pattern observed in mouse embryos, where expression of SDF-1 transcripts is prominent in several organ systems, including the developing neuronal, craniofacial, and cardiac systems (50).

Studies of mice lacking either SDF-1 (21) or CXCR4 (22, 23) revealed that the CXCR4/SDF-1 receptor/ligand system may be involved in cardiac ventricular septum formation. In the mouse embryo, CXCR4 mRNA is found in the aortopulmonary septum, whereas SDF-1 mRNA is specifically present in the outflow track and, to a lower extent, in the ventricular wall of the heart (51). This observation led the authors to suggest that the ventricular septum defect in SDF-1-/- or CXCR4-/- mice is caused by an interruption of SDF-1 signaling regulating the migration of the aortopulmonary septum during conotruncal development. In Xenopus embryos, CXCR4 mRNA appears to be absent from the heart anlage, but present in neural crest cells (25), which is just opposite to the distribution of SDF-1 mRNA reported here. Interestingly, neural crest cells originating from the caudal hindbrain migrate into the caudal pharyngeal arches, and a subset continues to migrate into the cardiac outflow tract where it will organize the outflow septum and form cholinergic cardiac ganglia of the parasympathic plexus. If these cells, also referred to as cardiac neural crest cells, are removed from the chick embryo before migration, several defects of heart and great arteries are observed, including persisting truncus arteriosus, overriding aorta, variable regression of the great arteries, and ventricular septum defect (52). Although it is currently unclear to what extent neural crest cells contribute to heart development in amphibia, it appears possible that the absence of SDF-1 from the heart anlage or of CXCR4 from cardiac neural crest cells causes a disturbance of the neural crest cell/cardiac myocyte interaction, which is required not only for normal septation of the cardiac outflow tract, but also for normal differentiation and function of the myocardium during early heart development (52).


   Acknowledgments
 
We thank Stephan Rütten, Ingrid Büsselmann, Karin Dillinger, and Susanne Gierschik for expert technical assistance.


   Footnotes
 
1 This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Medical Faculty of the University of Ulm. Back

2 Address correspondence and reprint requests to Dr. Peter Gierschik, Department of Pharmacology and Toxicology, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany. E-mail address: peter.gierschik{at}medizin.uni-ulm.de Back

3 Abbreviations used in this paper: SDF-1, stromal cell-derived factor 1; Sf9 cells, Spodoptera frugiperda cells; [35S]GTP[S], [35S]guanosine 5'-O-(3-thiotriphosphate); xSDF-1, Xenopus SDF-1; xCXCR4, Xenopus laevis CXCR4; h, human; MALDI-MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; GRO, growth-related oncogene; cSDF-1, cat SDF-1; mSDF-1, mouse SDF-1. Back

Received for publication August 16, 2001. Accepted for publication December 18, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Baggiolini, M., B. Dewald, B. Moser. 1997. Human chemokines: an update. Annu. Rev. Immunol. 15:675.[Medline]
  2. Zlotnik, A., O. Yoshie. 2000. Chemokines: a new classification system and their role in immunity. Immunity. 12:121.[Medline]
  3. Rossi, D., A. Zlotnik. 2000. The biology of chemokines and their receptors. Annu. Rev. Immunol. 18:217.[Medline]
  4. Locati, M., P. M. Murphy. 1999. Chemokines and chemokine receptors: biology and clinical relevance in inflammation and AIDS. Annu. Rev. Med. 50:425.[Medline]
  5. Broxmeyer, H. E., C. H. Kim. 1999. Regulation of hematopoiesis in a sea of chemokine family members with a plethora of redundant activities. Exp. Hematol. 27:1113.[Medline]
  6. Mackay, C. R.. 2001. Chemokines: immunology’s high impact factors. Nat. Immunol. 2:95.[Medline]
  7. Youn, B. S., C. Mantel, H. E. Broxmeyer. 2000. Chemokines, chemokine receptors and hematopoiesis. Immunol. Rev. 177:150.[Medline]
  8. Berger, E. A., P. M. Murphy, J. M. Farber. 1999. Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu. Rev. Immunol. 17:657.[Medline]
  9. Hesselgesser, J., R. Horuk. 1999. Chemokine and chemokine receptor expression in the central nervous system. J. Neurovirol. 5:13.[Medline]
  10. Murdoch, C., A. Finn. 2000. Chemokine receptors and their role in inflammation and infectious diseases. Blood. 95:3032.[Abstract/Free Full Text]
  11. Nagasawa, T., H. Kikutani, T. Kishimoto. 1994. Molecular cloning and structure of a pre-B-cell growth-stimulating factor. Proc. Natl. Acad. Sci. USA 91:2305.[Abstract/Free Full Text]
  12. Bleul, C. C., R. C. Fuhlbrigge, J. M. Casasnovas, A. Aiuti, T. A. Springer. 1996. A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1). J. Exp. Med. 184:1101.[Abstract/Free Full Text]
  13. Tashiro, K., H. Tada, R. Heilker, M. Shirozu, T. Nakano, T. Honjo. 1993. Signal sequence trap: a cloning strategy for secreted proteins and type I membrane proteins. Science 261:600.[Abstract/Free Full Text]
  14. Jiang, W., P. Zhou, S. M. Kahn, N. Tomita, M. D. Johnson, I. B. Weinstein. 1994. Molecular cloning of TPAR1, a gene whose expression is repressed by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). Exp. Cell. Res. 215:284.[Medline]
  15. Shirozu, M., T. Nakano, J. Inazawa, K. Tashiro, H. Tada, T. Shinohara, T. Honjo. 1995. Structure and chromosomal localization of the human stromal cell-derived factor 1 (SDF1) gene. Genomics. 28:495.[Medline]
  16. Nishimura, Y., T. Miyazawa, Y. Ikeda, Y. Izumiya, K. Nakamura, J. S. Cai, E. Sato, M. Kohmoto, T. Mikami. 1998. Molecular cloning and sequencing of feline stromal cell-derived factor-1{alpha} and {beta}. Eur. J. Immunogenet. 25:303.[Medline]
  17. Gleichmann, M., C. Gillen, M. Czardybon, F. Bosse, R. Greiner-Petter, J. Auer, H. W. Muller. 2000. Cloning and characterization of SDF-1{gamma}, a novel SDF-1 chemokine transcript with developmentally regulated expression in the nervous system. Eur. J. Neurosci. 12:1857.[Medline]
  18. Nagasawa, T., K. Tachibana, T. Kishimoto. 1998. A novel CXC chemokine PBSF/SDF-1 and its receptor CXCR4: their functions in development, hematopoiesis and HIV infection. Semin. Immunol. 10:179.[Medline]
  19. Murphy, P. M., M. Baggiolini, I. F. Charo, C. A. Hebert, R. Horuk, K. Matsushima, L. H. Miller, J. J. Oppenheim, C. A. Power. 2000. International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol. Rev. 52:145.[Abstract/Free Full Text]
  20. Murdoch, C.. 2000. CXCR4: chemokine receptor extraordinaire. Immunol. Rev. 177:175.[Medline]
  21. Nagasawa, T., S. Hirota, K. Tachibana, N. Takakura, S. Nishikawa, Y. Kitamura, N. Yoshida, H. Kikutani, T. Kishimoto. 1996. Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 382:635.[Medline]
  22. Tachibana, K., S. Hirota, H. Iizasa, H. Yoshida, Y. Kawabata, Y. Kataoka, Y. Kitamura, K. Matsushima, N. Yoshida, S. Nishikawa, et al 1998. The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract. Nature 393:591.[Medline]
  23. Zou, Y.-R., A. H. Kottmann, M. Kuroda, I. Taniuchi, D. R. Littman. 1998. Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 393:595.[Medline]
  24. Ma, Q., D. Jones, P. R. Borghesani, R. A. Segal, T. Nagasawa, T. Kishimoto, R. T. Bronson, T. A. Springer. 1998. Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc. Natl. Acad. Sci. USA 95:9448.[Abstract/Free Full Text]
  25. Moepps, B., M. Braun, K. Knopfle, K. Dillinger, W. Knochel, P. Gierschik. 2000. Characterization of a Xenopus laevis CXC chemokine receptor 4: implications for hematopoietic cell development in the vertebrate embryo. Eur. J. Immunol. 30:2924.[Medline]
  26. Kardong, K. V.. 1998. Vertebrates: Comparative Anatomy, Function, Evolution 2nd Ed. WCB/McGraw-Hill, Boston.
  27. Ueda, H., M. A. Siani, W. Gong, D. A. Thompson, G. G. Brown, J. M. Wang. 1997. Chemically synthesized SDF-1a analogue, N33A, is a potent chemotactic agent for CXCR4/fusin/LESTR-expressing human leukocytes. J. Biol. Chem. 272:24966.[Abstract/Free Full Text]
  28. Kaufmann, E., H. Paul, H. Friedle, A. Metz, M. Scheucher, J. H. Clement, W. Knochel. 1996. Antagonistic actions of activin A and BMP-2/4 control dorsal lip-specific activation of the early response gene XFD-1' in Xenopus laevis embryos. EMBO J. 15:6739.[Medline]
  29. Nieuwkoop, P. D., J. Faber. 1994. Normal Table of Xenopus laevis (DAUDIN): A Systematical and Chronological Survey of the Development from the Fertilized Egg till the End of Metamorphosis Garland, New York.
  30. Moepps, B., R. Frodl, H.-R. Rodewald, M. Baggiolini, P. Gierschik. 1997. Two murine homologues of the human chemokine receptor CXCR4 mediating stromal cell-derived factor 1{alpha} activation of Gi2 are differentially expressed in vivo. Eur. J. Immunol. 27:2102.[Medline]
  31. Harland, R. M.. 1991. In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell Biol. 36:685.[Medline]
  32. Sambrook, J., E. F. Fritsch, T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
  33. Church, M. G., W. Gilbert. 1984. Genomic sequencing. Proc. Natl. Acad. Sci. USA 81:1991.[Abstract/Free Full Text]
  34. Bradford, M. M.. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248.[Medline]
  35. Oquendo, P., J. Alberta, D. Z. Wen, J. L. Graycar, R. Derynck, C. D. Stiles. 1989. The platelet-derived growth factor-inducible KC gene encodes a secretory protein related to platelet {alpha}-granule proteins. J. Biol. Chem. 264:4133.[Abstract/Free Full Text]
  36. von Heijne, G.. 1986. A new method for predicting signal sequence cleavage sites. Nucleic Acid Res. 14:4683.[Abstract/Free Full Text]
  37. Kenakin, T.. 1995. Agonist-receptor efficacy. II. Agonist trafficking of receptor signals. Trends Pharmacol. Sci. 16:232.[Medline]
  38. Hall, D. A., I. J. Beresford, C. Browning, H. Giles. 1999. Signaling by CXC-chemokine receptors 1 and 2 expressed in CHO cells: a comparison of calcium mobilization, inhibition of adenylyl cyclase and stimulation of GTP{gamma}S binding induced by IL-8 and GRO{alpha}. Br. J. Pharmacol. 126:810.[Medline]
  39. Zhang, S., B. S. Youn, J. L. Gao, P. M. Murphy, B. S. Kwon. 1999. Differential effects of leukotactin-1 and macrophage inflammatory protein-1{alpha} on neutrophils mediated by CCR1. J. Immunol. 162:4938.[Abstract/Free Full Text]
  40. Watson, C., G. Chen, P. Irving, J. Way, W. J. Chen, T. Kenakin. 2000. The use of stimulus-biased assay systems to detect agonist-specific receptor active states: implications for the trafficking of receptor stimulus by agonists. Mol. Pharmacol. 58:1230.[Abstract/Free Full Text]
  41. Dealwis, C., E. J. Fernandez, D. A. Thompson, R. J. Simon, M. A. Siani, E. Lolis. 1998. Crystal structure of chemically synthesized [N33A] stromal cell-derived factor 1{alpha}, a potent ligand for the HIV-1 "fusin" coreceptor. Proc. Natl. Acad. Sci. USA 95:6941.[Abstract/Free Full Text]
  42. Crump, M. P., J.-H. Gong, P. Loetscher, K. Rajarathnam, A. Amara, F. Arenzana-Seisdedos, J.-L. Virelizier, M. Baggiolini, B. D. Sykes, I. Clark-Lewis. 1997. Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1. EMBO J. 16:6996.[Medline]
  43. Siciliano, S. J., T. E. Rollins, J. A. DeMartino, Z. Konteatis, L. Malkowitz, G. Van Riper, S. Bondy, H. Rosen, M. S. Springer. 1994. Two-site binding of C5a by its receptor: an alternative binding paradigm for G protein-coupled receptors. Proc. Natl. Acad. Sci. USA 91:1214.[Abstract/Free Full Text]
  44. Elisseeva, E. L., C. M. Slupsky, M. P. Crump, I. Clark-Lewis, B. D. Sykes. 2000. NMR studies of active N-terminal peptides of stromal cell-derived factor-1: structural basis for receptor binding. J. Biol. Chem. 275:26799.[Abstract/Free Full Text]
  45. Loetscher, P., J.-H. Gong, B. Dewald, M. Baggiolini, I. Clark-Lewis. 1998. N-terminal peptides of stromal cell-derived factor-1 with CXC chemokine receptor 4 agonist and antiagonist activities. J. Biol. Chem. 273:22279.[Abstract/Free Full Text]
  46. Heveker, N., M. Montes, L. Germeroth, A. Amara, A. Trautmann, M. Alizon, J. Schneider-Mergener. 1998. Dissociation of the signaling and antiviral properties of SDF-1-derived small peptides. Curr. Biol. 8:369.[Medline]
  47. Amara, A., O. Lorthioir, A. Valenzuela, A. Magerus, M. Thelen, M. Montes, J. L. Virelizier, M. Delepierre, F. Baleux, H. Lortat-Jacob, F. Arenzana-Seisdedos. 1999. Stromal cell-derived factor-1{alpha} associates with heparan sulfates through the first {beta}-strand of the chemokine. J. Biol. Chem. 274:23916.[Abstract/Free Full Text]
  48. Sadir, R., F. Baleux, A. Grosdidier, A. Imberty, H. Lortat-Jacob. 2001. Characterization of the stromal cell-derived factor-1{alpha}/heparin complex. J. Biol. Chem. 276:8288.[Abstract/Free Full Text]
  49. Hansen, J. D., A. G. Zapata. 1998. Lymphocyte development in fish and amphibians. Immunol. Rev. 166:199.[Medline]
  50. McGrath, K. E., A. D. Koniski, K. M. Maltby, J. K. McGann, J. Palis. 1999. Embryonic expression and function of the chemokine SDF-1 and its receptor, CXCR4. Dev. Biol. 213:442.[Medline]
  51. Creazzo, T. L., R. E. Godt, L. Leatherbury, S. J. Conway, M. L. Kirby. 1998. Role of cardiac neural crest cells in cardiovascular development. Annu. Rev. Physiol. 60:267.[Medline]
  52. Waldo, K., M. Zdanowicz, J. Burch, D. H. Kumiski, H. A. Stadt, R. E. Godt, T. L. Creazzo, M. L. Kirby. 1999. A novel role for cardiac neural crest in heart development. J. Clin. Invest. 103:1499.[Medline]
  53. Thompson, J. D., D. G. Higgins, T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673.[Abstract/Free Full Text]
  54. Speicher, D. W. 2000. Mass spectrometry. In Current Protocols in Protein Science. E. J. Coligan, B. M. Dunn, D. W. Speicher, and P. T. Wingfield, eds. Wiley, New York, p. 16.1.15.



This article has been cited by other articles:


Home page
 J Mol EndocrinolHome page
R. Stumm and V. Hollt
CXC chemokine receptor 4 regulates neuronal migration and axonal pathfinding in the developing nervous system: implications for neuronal regeneration in the adult brain
J. Mol. Endocrinol., March 1, 2007; 38(3): 377 - 382.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Suzuki, Y. Takamura, M. Maeno, S. Tochinai, D. Iyaguchi, I. Tanaka, J. Nishihira, and T. Ishibashi
Xenopus laevis Macrophage Migration Inhibitory Factor Is Essential for Axis Formation and Neural Development
J. Biol. Chem., May 14, 2004; 279(20): 21406 - 21414.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Braun, M.
Right arrow Articles by Moepps, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Braun, M.
Right arrow Articles by Moepps, B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS