Combined phylogenetic and chromosomal location studies suggest that the orphan receptor RDC1 is related to CXC chemokine receptors. RDC1 provides a co-receptor function for a restricted number of human immunodeficiency virus (HIV) isolates, in particular for the CXCR4-using HIV-2 ROD strain. Here we show that CXCL12, the only known natural ligand for CXCR4, binds to and signals through RDC1. We demonstrate that RDC1 is expressed in T lymphocytes and that CXCL12-promoted chemotaxis is inhibited by an anti-RDC1 monoclonal antibody. Concomitant blockade of RDC1 and CXCR4 produced additive inhibitory effects in CXCL12-induced T cell migration. Furthermore, we provide evidence that interaction of CXCL12 with RDC1 is specific, saturable, and of high affinity (apparent K D ≈ 0.4 nM). In CXCR4-negative cells expressing RDC1, CXCL12 promotes internalization of the receptor and chemotactic signals through RDC1. Collectively, our data indicate that RDC1, which we propose to rename as CXCR7, is a receptor for CXCL12.

The important family of G protein-coupled receptors has so far not been targeted very successfully with conventional monoclonal antibodies. Here we report the isolation and characterization of functional VHH-based immunoglobulin single variable domains (or nanobodies) against the chemokine receptor CXCR4. Two highly selective monovalent nanobodies, 238D2 and 238D4, were obtained using a time-efficient whole cell immunization, phage display, and counterselection method. The highly selective VHH-based immunoglobulin single variable domains competitively inhibited the CXCR4-mediated signaling and antagonized the chemoattractant effect of the CXCR4 ligand CXCL12. Epitope mapping showed that the two nanobodies bind to distinct but partially overlapping sites in the extracellular loops. Short peptide linkage of 238D2 with 238D4 resulted in significantly increased affinity for CXCR4 and picomolar activity in antichemotactic assays. Interestingly, the monovalent nanobodies behaved as neutral antagonists, whereas the biparatopic nanobodies acted as inverse agonists at the constitutively active CXCR4-N3.35A. The CXCR4 nanobodies displayed strong antiretroviral activity against T cell-tropic and dual-tropic HIV-1 strains. Moreover, the biparatopic nanobody effectively mobilized CD34-positive stem cells in cynomolgus monkeys. Thus, the nanobody platform may be highly effective at generating extremely potent and selective G protein-coupled receptor modulators.nanobody | CXCR4 antagonist | chemotaxis | HIV | stem cell mobilization

WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome is an immune deficiency linked in many cases to heterozygous mutations causing truncations in the cytoplasmic tail of CXC chemokine receptor 4 (CXCR4). Leukocytes expressing truncated CXCR4 display enhanced responses to the receptor ligand CXCL12, including chemotaxis, which likely impair their trafficking and contribute to the immunohematologic clinical manifestations of the syndrome. CXCR4 desensitization and endocytosis are dependent on ␤-arrestin (␤arr) recruitment to the cytoplasmic tail, so that the truncated CXCR4 are refractory to these processes and so have enhanced G protein-dependent signaling. Here, we show that the augmented responsiveness of WHIM leukocytes is also accounted for by enhanced ␤arr2-dependent signaling downstream of the truncated CXCR4 receptor. Indeed, the WHIM-associated receptor CXCR4 1013 maintains association with ␤arr2 and triggers augmented and prolonged ␤arr2-dependent signaling, as revealed by ERK1/2 phosphorylation kinetics. Evidence is also provided that CXCR4 1013 -mediated chemotaxis critically requires ␤arr2, and disrupting the SHSK motif in the third intracellular loop of CXCR4 1013 abrogates ␤arr2-mediated signaling, but not coupling to G proteins, and normalizes chemotaxis. We also demonstrate that CXCR4 1013 spontaneously forms heterodimers with wild-type CXCR4. Accordingly, we propose a model where enhanced functional interactions between ␤arr2 and receptor dimers account for the altered responsiveness of WHIM leukocytes to CXCL12. (Blood. 2008;112:34-44) IntroductionThe G-protein-coupled receptor (GPCR) CXC chemokine receptor 4 (CXCR4) and its ligand, the stromal cell-derived factor-1 (CXCL12/SDF-1), regulate leukocyte hematopoiesis and trafficking. 1 They initiate signal transduction by activating heterotrimeric G␣␤␥-proteins, which then act on effectors to trigger downstream cellular responses. 2 CXCL12 also elicits processes causing receptor desensitization, which results in the uncoupling from G-proteins and involves phosphorylation of the CXCR4 cytoplasmic tail (C-tail) by protein kinase C and GPCR kinases (GRKs) and interaction of the phosphorylated receptor with ␤-arrestins (␤arrs). [3][4][5] ␤arrs then target desensitized CXCR4 to clathrin-coated pits for endocytosis. ␤arrs also link GPCRs to the stimulation of additional signaling molecules, including members of the mitogen-activated protein kinase (MAPK) family. 6 Studies on CXCR4 have demonstrated that ␤-arrestin2 (␤arr2) strengthens activation of the p38 and extracellular signal-regulated kinase (ERK) MAPKs and CXCL12-induced chemotaxis. 5,7,8 WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome (WS) is a rare immunodeficiency disease linked to CXCR4 dysfunctions and is characterized by warts, recurrent bacterial infections, hypogammaglobulinemia, lymphopenia, and myelokathexis, a severe neutropenia with abnormal retention of mature neutrophils in the bone marrow (BM). 9,10 WS, most often inherited as an a...

The CXCL12γ chemokine arises by alternative splicing from Cxcl12, an essential gene during development. This protein binds CXCR4 and displays an exceptional degree of conservation (99%) in mammals. CXCL12γ is formed by a protein core shared by all CXCL12 isoforms, extended by a highly cationic carboxy-terminal (C-ter) domain that encompass four overlapped BBXB heparan sulfate (HS)-binding motifs. We hypothesize that this unusual domain could critically determine the biological properties of CXCL12γ through its interaction to, and regulation by extracellular glycosaminoglycans (GAG) and HS in particular. By both RT-PCR and immunohistochemistry, we mapped the localization of CXCL12γ both in mouse and human tissues, where it showed discrete differential expression. As an unprecedented feature among chemokines, the secreted CXCL12γ strongly interacted with cell membrane GAG, thus remaining mostly adsorbed on the plasmatic membrane upon secretion. Affinity chromatography and surface plasmon resonance allowed us to determine for CXCL12γ one of the higher affinity for HS (Kd = 0.9 nM) ever reported for a protein. This property relies in the presence of four canonical HS-binding sites located at the C-ter domain but requires the collaboration of a HS-binding site located in the core of the protein. Interestingly, and despite reduced agonist potency on CXCR4, the sustained binding of CXCL12γ to HS enabled it to promote in vivo intraperitoneal leukocyte accumulation and angiogenesis in matrigel plugs with much higher efficiency than CXCL12α. In good agreement, mutant CXCL12γ chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment. We conclude that CXCL12γ features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain. The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12γ the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells.