CXCR4

CXCR4 Bernhard Moser* Theodor-Kocher Institute, University of Bern, Freiestrasse 1, Bern, 3012, Switzerland * corresponding author tel: 41-31-631-4157, fax: 41-31-631-3799, e-mail: [email protected] DOI: 10.1006/rwcy.2000.21004.

SUMMARY

BACKGROUND

CXCR4 was cloned on the basis of structural homology with other chemokine receptors and on the basis of its coreceptor activity in an HIV-1 fusion assay. The receptor selectively interacts with a single CXC chemokine, SDF-1. An unusual feature of this chemokine receptor is its broad range of cellular distribution with high expression on immature and mature hematopoietic cells as well as many tissue cells, including endothelial cells, neurons, and astrocytes. Freshly isolated peripheral blood T lymphocytes express high levels of CXCR4, most of which is stored in intracellular compartments, and cell culture under stimulatory conditions reduces CXCR4 gene expression. Short-term activation (e.g. via TCR-triggering) or exposure to SDF-1 leads to rapid CXCR4 phosphorylation and receptor internalization. CXCR4 plays a critical role in embryogenesis, as demonstrated in CXCR4- or SDF-1-deficient mice which die at late embryonic stages and show severe defects in myelo- and B lymphopoiesis, brain and heart organogenesis, and blood vessel formation in the intestinal tract. The function of CXCR4 (and its ligand) in adult blood and tissue cells is not known but some reports suggest a role in the localization of hematopoietic progenitor cells in the bone marrow and the maturation of platelets during transendothelial migration of megakaryocytes. CXCR4 is one of the two major HIV-1/2 coreceptors, characterizing CXCR4-dependent (X4) viruses which tend to appear at late stages in infected individuals. The envelope glycoproteins gp120 of X4 viruses act as a surrogate ligand for CXCR4 and mediate monocyte chemotaxis and apoptosis in CD8+ T lymphocytes (via macrophage activation) and neurons.

Discovery Several groups cloned the cDNA for human CXCR4 in the early 1990s by hybridization screening of cDNA libraries with DNA probes corresponding to the rabbit IL-8 receptor (Loetscher et al., 1994), the bovine neuropeptide Y receptor (Herzog et al., 1993; Jazin et al., 1993), or by PCR amplification using degenerate oligonucleotide primers corresponding to chemokine/chemoattractant receptors (Federsppiel et al., 1993; Nomura et al., 1993).

Alternative names Prior to identification of SDF-1 as the selective ligand for this receptor, CXCR4 was an orphan receptor termed LESTR (Loetscher et al., 1994), pBE1.3/ HUMSTR (Federsppiel et al., 1993), HM89 (Nomura et al., 1993), hFB22 (Jazin et al., 1993) and hL5R (Herzog et al., 1993).

Structure CXCR4 is a typical chemokine receptor with seven transmembrane-spanning domains which couples to heterotrimeric G proteins for signal transduction.

Main activities and pathophysiological roles Signaling through CXCR4 induces chemotactic migration and adhesion of receptor-bearing cells to

2010 Bernhard Moser integrin ligands, as is typical for chemokine receptors. However, unlike other chemokine receptors, CXCR4 is expressed on all types of leukocytes and a role of this receptor in the steady-state turnover and homing of blood leukocytes is suggested. In contrast to chemokine receptors for inducible chemokines which are produced in inflammatory conditions, CXCR4 does not regulate leukocyte traffic to sites of infection and disease. In addition, CXCR4 is present on hematopoietic progenitor cells in the bone marrow and thymus and its ligand SDF-1 mobilizes CD34+ progenitor cells. CXCR4 and SDF-1 have additional functions that are unrelated to leukocyte chemotaxis. An essential role in embryogenesis was demonstrated in studies of mouse embryos that were genetically engineered to be deficient in SDF-1 or CXCR4. Deletion of either gene was lethal before or shortly after birth, and mutant embryos showed severe defects in B cell development and myelopoiesis, and had defective ventricular septum formation of the heart, impaired cerebellar development, and abnormal mesenteric blood vessel formation. CXCR4 is the only entry cofactor for syncytium-inducing HIV particles which typically emerge at the onset of AIDS, and rapid progression of this disease may be due to the unusually wide distribution of CXCR4 in blood and tissue cells. CXCR4 antagonists may represent a novel strategy for the treatment of patients with AIDS.

GENE

Accession numbers The human LESTR/CXCR4 cDNA of 1645 bp has the GenBank/EMBL databank accession number X71635 (Loetscher et al., 1994). Ortholog receptor cDNAs are described in chimpanzee (U89797), rhesus monkey (U73740), baboon (AF031089), African green monkey (AB015943), cow (M86739), and mouse (D87747) which express alternatively spliced mRNAs for two CXCR4 isoforms (Nagasawa et al., 1996b; Heesen et al., 1997; Moepps et al., 1997), rat (U90610), cat (U63558), and fish (AB012310). Sequences of the human and murine CXCR4 genes are deposited under Y14739, AJ224869, and X99581, respectively.

Chromosome location and linkages The human and murine CXCR4 genes are localized on human chromosome 2q21 and mouse chromosome 1.

PROTEIN

Sequence Protein sequence information can be retrieved from the GenBank/EMBL databank entries (see Gene: Accession numbers).

Description of protein Human CXCR4 is a membrane-spanning glycoprotein of 352 amino acids (Mr 39,745). CXCR4 has seven transmembrane helices, which is typical for chemokine receptors (Loetscher et al., 1994). CXCR4 shares with other chemokine receptors the DRYLAIVHA motif in the second intracellular loop and two Cys residues in the N-terminal region and the third extracellular loop, which may form an essential disulfide bond. Two potential N-glycosylation sites are located in the N-terminal region (Asn11-Tyr-Thr) and the second extracellular loop (Asn177-Val-Ser). CXCR4 shares several putative substrate motifs for protein kinase C with other chemokine receptors. The intracellular C-terminal region contains 18 Ser/Thr residues, some of which are involved in SDF-1-induced CXCR4 internalization through phosphorylation by receptor-specific kinases and/or Ser/Thr protein kinases (Amara et al., 1997; Haribabu et al., 1997).

Relevant homologies and species differences Overall, amino acid sequence identity with other chemokine receptors is approximately 30%. Highest similarity is found in the transmembrane domains whereas the extracellular N-terminal region and the intracellular C-terminal region are less well conserved. CXCR4 couples to the class of G proteins which are sensitive to Bordetella pertussis toxin. CXCR4 is a member of the superfamily of G proteincoupled receptors which recognize ligands as varied as hormones, neurotransmitters, lipid derivatives, bacterial cell wall components, odorants, and light. The sequences of human and African green monkey CXCR4 differ in only four amino acids, resulting in an amino acid sequence identity of 98.6%. Sequence conservation is further documented by 91.2% identity between human and murine CXCR4, and fish CXCR4 which shares 64.2% amino acids with the human receptor. The sequence of its ligand SDF-1 is equally well conserved, as documented by the single conservative

CXCR4 2011 amino acid difference between the human and mouse form.

Affinity for ligand(s) The two isoforms SDF-1 and SDF-1 bind to CXCR4 with a Kd of 2±9 nM, as assessed in [125I]SDF-1 binding studies using CEM cells (Crump et al., 1997; Loetscher et al., 1998). Numerous structural variants of SDF-1 exist which bind to CXCR4 with lower affinity (Crump et al., 1997; Heveker et al., 1998; Loetscher et al., 1998).

Cell types and tissues expressing the receptor CXCR4 is widely expressed in blood and tissue cells, which is highly unusual for chemokine receptors (Table 1). The expression and function of CXCR4 in neutrophils is controversial. Some reports describe CXCR4 expression, by mRNA and protein analysis, and chemotactic responses to SDF-1 (Loetscher et al., 1994; Oberlin et al., 1996; Foerster et al., 1998), whereas no activity was reported by others (Bleul et al., 1996b, 1997; Hori et al., 1998). Donor-to-donor variations in the neutrophil preparations may explain the observed differences. Alternatively, the antibodies that were used may have differed in their capacity to detect cell surface CXCR4. All reports agree that peripheral blood monocytes, T, and B lymphocytes are positive for CXCR4 and respond to SDF-1. In addition, CXCR4 is prominently expressed on hematopoietic progenitor cells and tissue cells, notably endothelial cells.

Regulation of receptor expression The recognition of CXCR4 as an HIV entry coreceptor has mobilized many laboratories to investigate the regulation of its expression in CD4+ cells, notably T lymphocytes. Flow cytometry showed that only about 20% of freshly isolated blood lymphocytes expressed cell surface CXCR4 whereas the majority (> 80%) of these cells stained positive for intracellular CXCR4 (Bermejo et al., 1998; Foerster et al., 1998). Interestingly, short-term culture in the absence of T cell stimuli resulted in the relocation of CXCR4 to the cell surface. These findings are in striking contrast to chemokine receptors for inducible chemokines which are absent in resting T lymphocytes and are induced upon cell culture in the presence

of IL-2 (Baggiolini, 1998; Moser et al., 1998). Bermejo and colleagues did not find preferential expression of CXCR4 on certain T cell subsets (Bermejo et al., 1998), whereas Bleul and colleagues (1997) reported predominant expression in resting naõÈ ve CD45RA+ T cells. Stimulation with PHA, IL-2, or a combination of anti-CD3 and anti-CD28 antibodies enhanced CXCR4 mRNA expression transiently, with peak values within days 3±6 of culture (Bleul et al., 1997; Carroll et al., 1997), mimicking the pattern of expression of the homing chemokine receptor CCR7 in T lymphocytes (Willimann et al., 1998; Yoshida et al., 1998). In agreement with these observations, promoter studies of human CXCR4 gene revealed considerable induction by IL-2 or antibodies to CD3 and CD28 (Moriuchi et al., 1997). The marked baselevel expression seen in resting circulating T lymphocytes may be attributed to the putative nuclear respiratory factor-binding site NRF-1. In contrast, responses to inducible chemokines and expression of corresponding chemokine receptors by cultured T cells are rapidly inhibited by anti-CD3 treatment (Loetscher et al., 1996; Carroll et al., 1997), indicating that signaling through the T cell receptor differentially regulates expression of the two classes of proinflammatory and homing types of chemokine receptors. IL-4 and TGF were also found to induce CXCR4 expression, whereas IFN was inhibitory, as shown for T cells, monocytes, dendritic cells, and endothelial cells (Gupta et al., 1998; Jourdan et al., 1998; Penton-Rol et al., 1998; Zella et al., 1998; Zoeteweij et al., 1998). In addition, cultured endothelial cells express increased levels of CXCR4 after treatment with basic FGF, which is prevented by TNF (Feil and Augustin, 1998). In addition to regulation of gene expression by cytokines and other stimuli, the level of cell surface CXCR4 is rapidly modulated during T cell activation, as shown by short-term treatment (up to 120 minutes) with phytohemagglutinin, anti-CD3, or PMA which resulted in depletion of cell surface CXCR4 (Amara et al., 1997; Haribabu et al., 1997; Signoret et al., 1997; Bermejo et al., 1998; Foerster et al., 1998; Jourdan et al., 1998). This effect was probably due to Ser/The kinase-dependent receptor phosphorylation which induced receptor internalization through coated pits/ vesicles and localization in endosomal compartments (Haribabu et al., 1997; Signoret et al., 1997). CXCR4 internalization was also rapidly achieved by addition of SDF-1, which was independent of signaling by B. pertussis toxin-sensitive G proteins, but involved the intracellular C-terminal region of the receptor (Amara et al., 1997; Foerster et al., 1998; Tarasova et al., 1998). Removal of SDF-1 restored cell surface levels of CXCR4, indicating that local production of

Table 1 Expression of CXCR4 in blood and tissue cells RNA Northerna

Protein In situb

Cell surfacec

References Tissued

Mature blood cells Neutrophils

+

+

Loetscher et al., 1994; Foerster et al., 1998

Monocytes

+

+

Loetscher et al., 1994; Bleul et al., 1997; Foerster et al., 1998; Hori et al., 1998; Ostrowski et al., 1998; Penton-Rol et al., 1998

Macrophages

+

+

Vallat et al., 1998; Zhang et al., 1998

Dendritic cells

+

+

Microglia

+

+

T lymphocytes

+

+

Loetscher et al., 1994; Bleul et al., 1997; Berkowitz et al., 1998; Bermejo et al., 1998; Foerster et al., 1998; Hori et al., 1998; Jourdan et al., 1998; Ostrowski et al., 1998; Zhang et al., 1998;

B lymphocytes

+

+

Loetscher et al., 1994; Bleul et al., 1997, 1998; Foerster et al., 1998; Hori et al., 1998

+

Hamada et al., 1998

+

Deichmann et al., 1997; MoÈhle, 1998

+

D'Apuzzo et al., 1997

Megakaryocytes/platelets

Granelli-Piperno et al., 1996; Ayehunie et al., 1997; Zoeteweij et al., 1998 +

Lavi et al., 1997; Tanabe et al., 1997; Vallat et al., 1998

Hematopoietic cells CD34ÿ cells Pro-/pre-B cells

+

Thymocytes

+

Kitchen and Zack, 1997; Moepps et al., 1997; Berkowitz et al., 1998; Kim et al., 1998; Zhang et al., 1998

Tissue cells Endothelial cells

+

Neurons

+

Astrocytes

+

+

+

+

Gupta et al., 1998; Tachibana et al., 1998; Feil and Augustin, 1998; Volin et al., 1998

+

Hesselgesser et al., 1997; Lavi et al., 1997; Meucci et al., 1998; Vallat et al., 1998

+

Tanabe et al., 1997

Tissues Liver

+

Colon

+

Brain

+

Heart

+

Federsppiel et al., 1993

Thymus

+

Nagasawa et al., 1996b; Moepps et al., 1997

Bone marrow

+

Moepps et al., 1997

Lymph node

+

Nagasawa et al., 1996b; Moepps et al., 1997

Spleen

+

Nagasawa et al., 1996b; Moepps et al., 1997

a

Federsppiel et al., 1993 Federsppiel et al., 1993 +

Federsppiel et al., 1993; Jazin, 1993; Lavi et al., 1997; Moepps et al., 1997; Zou et al., 1998;

CXCR4 mRNA in freshly isolated or cultured cells and tissues detected by northern blot or RT-PCR.

b

CXCR4 mRNA expression in tissue sections analyzed by in situ hybridization.

c

Flow cytometric detection of cell surface/intracellular CXCR4 protein.

d

CXCR4 protein in tissue sections analyzed by immunohistochemistry.

CXCR4 2013 this chemokine plays an important (negative-feedback) role in the regulation of T lymphocyte migration to SDF-1 and infection by CXCR4-tropic HIV particles. Finally, the envelope glycoprotein gp120 of X4 HIV-1 isolates was reported to inhibit chemokine responses in CD4-bearing cells through binding to and surface depletion of CD4 and CXCR4 (Madani et al., 1998; Wang et al., 1998).

Release of soluble receptors There is no report of soluble, secreted CXCR4 which is probably due to its serpentine-like membrane insertion.

SIGNAL TRANSDUCTION CXCR4, like all other chemokine receptors, couples to heterotrimeric G proteins (Gi or Gq subtype) and its activity is regulated by phosphorylation and subsequent internalization. SDF-1-mediated signaling leads to a range of cellular responses which can be divided into immediate responses (chemotactic migration involving cytoskeletal rearrangement and integrin-mediated adhesion) and long-term responses which depend on gene expression.

Associated or intrinsic kinases Signal transduction elements that become activated during SDF-1-mediated CXCR4 signaling include focal adhesion components and diverse kinases which are critical for chemokine receptor inactivation, cell migration and gene expression (Davis et al., 1997; Haribabu et al., 1997; Bermejo et al., 1998; Ganju et al., 1998; Jourdan et al., 1998). SDF-1 induces protein tyrosine phosphorylation of the related adhesion focal kinase RAFTK (also known as Pyk2), paxillin and the docking protein Crk which show enhanced association upon activation (Davis et al., 1997; Ganju et al., 1998). Similarly, phosphatidylinositol 3-kinase becomes rapidly phosphorylated, and its inhibition by wortmannin prevents paxillin phosphorylation and cell migration (Ganju et al., 1998).

Cytoplasmic signaling cascades Further downstream elements involved in transcriptional activation are p44/42 mitogen-activated protein (MAP) kinases Erk 1 and 2 which show enhanced phosphorylation upon SDF-1 stimulation (possibly

through MAP kinase kinase 1 activation), whereas p38 MAP kinase and JNK remain unaffected (Ganju et al., 1998; Jourdan et al., 1998).

DOWNSTREAM GENE ACTIVATION

Transcription factors activated CXCR4 signaling leads to activation of the nuclear transcriptional factor NFB which is critically involved in gene expression during stimulation of lymphocytes and other inflammatory cells as well as HIV replication (Ganju et al., 1998).

Genes induced The effect of chemokines on gene expression is not well understood. Yet, induction of membrane-bound TNF and TNF receptor type II expression during SDF-1-mediated apoptosis in T cells suggests that signaling through CXCR4 in macrophages and T cells resulted in expression of the corresponding genes (Herbein et al., 1998).

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors CXCR4 mediates chemotactic migration and rapid/ transient adhesion through integrin activation which are typical responses seen with chemokines in leukocytes. Of note, freshly isolated or short-term activated lymphocytes which do not respond to those chemokines known to be produced at sites of inflammation express high levels of CXCR4 and readily migrate to SDF-1 (Loetscher et al., 1994; Bleul et al., 1996a,b, 1997; Oberlin et al., 1996; Bermejo et al., 1998). An additional hallmark of CXCR4 is its wide expression in mature leukocytes present in blood and tissues (Table 1), which may be relevant for SDF-1-mediated localization of leukocytes at sites of steady-state turnover. The envelope glycoprotein gp120 of CXCR4tropic HIV particles acts as a second type of CXCR4 ligand and induces CD4+ T cell activation.

2014 Bernhard Moser CXCR4 may also be involved in the CD8+ T lymphocyte apoptosis generally observed during HIV infection (Davis et al., 1997; Herbein et al., 1998). This was confirmed in mixed cultures containing HIV-infected lymphocytes and macrophages in which the number of CD8+ T lymphocytes progressively diminished during viral propagation (Herbein et al., 1998). CXCR4-tropic HIV particles could be replaced by soluble gp120 or SDF-1, and apoptosis was attributed to induction of two genes, one for the membrane-bound TNF in macrophages and the other one for the TNF receptor type II in CD8+ T lymphocytes. Apoptosis in CD8+ T lymphocytes did not occur in the absence of macrophages. However, ligation of gp120 with CD4 and CXCR4 induced apoptosis in CD4+ T lymphocytes which was blocked by SDF-1, and in this case the mechanisms involved remain to be determined (Berndt et al., 1998). A report describes the direct induction of apoptosis in a human neuronal cell line by SDF-1 via CXCR4 (Hesselgesser et al., 1998), which is in contrast to another report demonstrating inhibition (rather than induction) of HIV envelope proteinmediated apoptosis in cultured hippocampal neurons by SDF-1 (Meucci et al., 1998). Numerous articles describe the expression of CXCR4 and activity of SDF-1 in myelo- and lymphopoietic progenitor cells (Nagasawa et al., 1994; Aiuti et al., 1997; D'Apuzzo et al., 1997; Deichmann et al., 1997; Kitchen and Zack, 1997; Kim et al., 1998; Berkowitz et al., 1998; MoÈhle et al., 1998). Originally, SDF-1 was identified on the basis of its co-mitogenic activity together with IL-7 for pre-B cells but this function has not been investigated in great detail (Nagasawa et al., 1994). Instead, SDF-1 was found to be a potent chemoattractant for early progenitor cells in the bone marrow, including CD34+ cells, and pre-/pro-B cells (Aiuti et al., 1997; D'Apuzzo et al., 1997). More developed B cell progenitors still expressed CXCR4 but lacked responsiveness, suggesting that SDF-1 acts on those hematopoietic progenitor cells which fully depend on contact with bone marrow stromal cells (D'Apuzzo et al., 1997). A similar situation is found in the thymus where highest CXCR4 expression and marked responses to SDF-1 were seen in the most immature thymocyte subsets, the triple-negative ( TCRÿCD4ÿCD8ÿ) and the double-positive (CD4+CD8+) thymocytes (Kitchen and Zack, 1997; Berkowitz et al., 1998; Kim et al., 1998). Also here, SDF-1 may have a trapping function by preventing egress of immature thymocytes from cortex to medulla. SDF-1/CXCR4 may also play an important role in platelet formation. Mature megakaryocytes were shown to migrate in response to SDF-1 through cultured bone marrow endothelial cell monolayers,

and this process resulted in the release of platelets which expressed CXCR4 (Hamada et al., 1998). Using an elegant cDNA cloning strategy, Feng and colleagues have identified CXCR4 (termed fusin by these authors) as entry cofactor, which complemented CD4 on target cells during fusion with syncytiuminducing HIV-1 strains (Feng et al., 1996). This finding demonstrated for the first time that chemokine receptors are critically involved in HIV infection. Today, it is clear that the distribution of CXCR4 and other chemokine receptors with cofactor function determines viral tropism and pathogenesis (Moore et al., 1997; Cairns and D'Souza, 1998). SDF-1 prevents entry of CXCR4-tropic (X4) HIV-1 strains through binding to CXCR4 on CD4+ T cells and consequent rapid internalization of ligand±receptor complexes (Bleul et al., 1996a; Oberlin et al., 1996; Amara et al., 1997). In contrast to CCR5-tropic (R5) viruses which predominate at asymptomatic stages in infected individuals, X4 viruses typically emerge during onset of AIDS (Moore et al., 1997; Cairns and D'Souza, 1998). Disease progression is accompanied by a change in coreceptor usage in emerging viruses from CCR5 to CXCR4, and the consequent rapid decline in immune functions may reflect the broad expression of CXCR4 in blood and tissue cells (Table 1). Of note, CXCR4 is also expressed in microglial cells, astrocytes, and neurons which may function as viral reservoirs during HIV infection and, in addition, may contribute to brain disorders (dementia) frequently observed in infected individuals (Hesselgesser et al., 1997; Lavi et al., 1997; Tanabe et al., 1997; Vallat et al., 1998). CXCR4 also functions as a CD4-independent receptor for HIV-2 (Endres et al., 1996). As with CCR5, much effort is being taken to develop synthetic inhibitors which selectively bind to CXCR4, and several reports describe the structure and function of such molecules (Baggiolini and Moser, 1997).

Phenotypes of receptor knockouts and receptor overexpression mice Mice deficient in either SDF-1 (Nagasawa et al., 1996a; Ma et al., 1998) or CXCR4 (Ma et al., 1998; Tachibana et al., 1998; Zou et al., 1998) have been generated by targeted disruption of the respective genes. Both gene knockout animals show identical phenotypes, indicating that SDF-1 is the primary physiologic ligand for CXCR4 and that other receptors for this chemokine do not exist. Whereas heterozygous (SDF-1+/ÿ, CXCR4+/ÿ) animals are healthy and fertile, homozygous disruption resulted

CXCR4 2015 in fetal mortality in SDF-1ÿ/ÿ and CXCR4ÿ/ÿ embryos. Mutant embryos were present at expected ratios up to day 15.5 of embryogenesis (E15.5), but half of the embryos were dead at E18.5 and neonates died within an hour after birth. Phenotypic aberrations include defects in hematopoiesis, heart and brain development, and vascularization of the gastrointestinal tract. SDF-1ÿ/ÿ and CXCR4ÿ/ÿ embryos show severely reduced B lymphopoiesis and myelopoiesis in the bone marrow, which is in agreement with expression of CXCR4 in early hematopoietic progenitor cells and their chemotactic responses to SDF-1 (Aiuti et al., 1997; D'Apuzzo et al., 1997; Deichmann et al., 1997). In the fetal liver, however, myelopoiesis was not affected and monocytes, macrophages, granulocytes, and megakaryocytes developed normally. Also, T lymphopoiesis was not affected in mutant embryos, indicating that thymocyte maturation does not depend on SDF-1/ CXCR4. All mutants showed defects in the ventricular septum formation of the heart. In CXCR4ÿ/ÿ embryos all major blood vessels were present. However, blood vessel branching, notably in the vasculature of the gastrointestinal tract, was severely reduced (Tachibana et al., 1998). This striking dependence on SDF-1/CXCR4 is supported by the recent evidence of CXCR4 expression in cultured endothelial cells and responses to SDF-1 (Feil and Augustin, 1998; Gupta et al., 1998; Tachibana et al., 1998; Volin et al., 1998). Similarly, the expression of CXCR4 in the brain suggests that SDF-1 and its receptor may play a role in brain development (Hesselgesser et al., 1997; Lavi et al., 1997; Moepps et al., 1997; Tanabe et al., 1997; Vallat et al., 1998) and this was confirmed by the observed defects in cerebellar anlage formation and abnormal granule cell localization in CXCR4ÿ/ÿ embryos (Zou et al., 1998).

THERAPEUTIC UTILITY

Effects of inhibitors (antibodies) to receptors Several low molecular weight compounds with selectivity for CXCR4 are described. The first is a heterocyclic bicyclam derivative, called AMD3100, which has been known for some time to inhibit the replication of T cell line-adapted (X4) HIV-1 isolates. The mechanism of action remained unknown until De Clercq and colleagues demonstrated a selective interaction of AMD3100 with CXCR4 (Schols et al.,

1997). It diminished binding of a specific antibody to cell surface CXCR4, possibly through induction of receptor internalization, and completely blocked SDF-1-induced Ca2+ responses at 100 ng/mL. Although nontoxic at high concentrations in mice, poor oral bioavailability makes AMD3100 an unlikely candidate drug for use in clinical trials. Two other inhibitors of CXCR4 are small peptides which, like SDF-1-derived peptides, are presently not considered useful for the treatment of AIDS. T22 is an 18 amino acid peptide from the hemocyte debris of American horseshoe crab which prevents infection by X4 HIV-1 isolates through blockage of CXCR4 on CD4+ target cells (Murakami et al., 1997). Similarly, the nona-D-arginine peptide ALX40-4C shows a high degree of selectivity for CXCR4 and blocked SDF-1-induced Ca2+ responses and infection by X4 HIV-1 strains at low micromolar concentrations (Doranz et al., 1997). Finally, a distamycin derivative (NSC 651016) was found to block infection by both CXCR4- and CCR5-dependent HIV-1 strains, which was probably due to coreceptor downmodulation (Howard et al., 1998). The use of monoclonal antibodies (mAbs) represents an alternative strategy for the treatment of HIV-infected individuals and several anti-CXCR4 mAbs with HIV-blocking activity are described. The first such mAb 12G5 was generated by Hoxie and colleagues who made it readily available to the scientific community (Endres et al., 1996; McKnight et al., 1997). Additional mAbs that block infection by X4 HIV-1 and HIV-2 isolates through binding to CXCR4 are 2B11 (Foerster et al., 1998), IVR7, and TSH123 (Hori et al., 1998).

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