This study provides extensive characterization of the novel high affinity SDF-1 (CXCL12)–binding receptor that we reported in an earlier publication (14
). The novel receptor, initially designated CCX CKR2 (14
) but renamed CXCR7 in this paper, is a 7-transmembrane receptor encoded by RDC1
), a gene that, before our initial report (14
), belonged to the family of orphan receptors with unknown ligands (20
). In addition to binding SDF-1, CXCR7 is also a high affinity receptor for I-TAC (CXCL11) that, before our investigations, was regarded as a ligand for CXCR3 only. Our data show that CXCR7 regulates several important biological processes including cell survival, cell adhesion, and tumor development in animal models. CXCR7 is expressed on many tumor cells but not on most nontransformed cells. Although not expressed on unstimulated endothelial cells, CXCR7 can be induced, in vitro, on cells that form the neovasculature, a finding that is consistent with the independent studies of Madden et al., who have observed up-regulation of RDC1 expression in neovasculature associated with malignant gliomas (23
). Some of the effects observed have been previously associated with SDF-1 activity but have been thought, perhaps erroneously, to be mediated entirely through CXCR4. The elucidation of CXCR7 may require a reexamination of much of the previous body of work that presumed a mutually exclusive biological interaction between SDF-1 and CXCR4. Indeed, suggestions of discrepant CXCR4 expression and SDF-1 responsiveness already exist in the literature. For example, previous studies demonstrate that adhesion of E11 fetal liver cells to bone marrow endothelium can be neutralized by anti–SDF-1 antibodies despite the fact that the E11 fetal liver cells do not migrate in response to SDF-1 (24
The gene that encodes CXCR7, RDC1
, was initially cloned from a dog thyroid cDNA library using degenerate PCR primers corresponding to consensus sequences in the transmembrane domains of known GPCR (21
). Subsequent isolation of human and mouse RDC1
) revealed >90% identity of both nucleotide and protein sequences of all three species, indicating an extremely high level of evolutionary conservation. RDC1 protein has been reported to be a vasoactive intestinal peptide receptor (26
) and an adrenomedulin receptor (27
), but these designations have fallen from general acceptance (28
- encoded CXCR7 is structurally similar to CXCR2, and the CXCR7 gene maps between those of CXCR2 and CXCR4 on mouse chromosome 1 (22
). It is likely that the RDC1/CXCR7
escaped earlier deorphanization and identification as a CXCR because it lacks certain typical and easily accessible functional properties of CXCRs, namely the ability to mediate chemotaxis and calcium mobilization after ligand binding. In this context, previous reports of CXCR4−/−
mice demonstrated the absence of SDF-1–induced functional responses such as chemotaxis (1
) but lacked binding experiments with radiolabeled SDF-1 that would have revealed the existence of the second SDF-1 receptor (i.e., CXCR7). Although numerous database search engines suggest that CXCR7/RDC1
is broadly expressed at the mRNA level (e.g., http://www.sagenet.org
; see also references 22
), our study demonstrates this is not the case at the level of membrane-associated CXCR7. This seeming discrepancy is explained by our direct demonstration of certain nontransformed cells that express CXCR7-specific mRNA but lack surface CXCR7 protein as measured by ligand binding assays or anti-CXCR7 antibody staining (). This observation potentially reflects some type of posttranslational regulation in CXCR7 expression. Although we have observed many examples in which nontransformed cells express CXCR7 mRNA but lack surface CXCR7, we have seen complete concordance to date in CXCR7 mRNA and surface CXCR7 expression in tumor cells, E13 mouse fetal liver cells, and activated endothelial cells (, , and ).
The absence of ligand-induced CXCR7-mediated calcium mobilization or cell migration suggests that the CXCR7 signaling pathway is distinct from the typical GPCR mechanism of other CXCRs. Although an alternative CXCR7-linked signal transduction pathway has not been identified in this manuscript, receptor-mediated signaling is implied by our observations that CXCR7 provides a growth/survival advantage and increased adhesiveness of cells ( and ). Indeed, preliminary evidence from microarray analyses suggests that CXCR7 may be constitutively active in tumor cell lines, thereby resembling numerous constitutively active non-CXCR GPCRs (31
), as well as some virally encoded CXCRs; e.g., CMV-encoded US28 (34
). The same studies (31
) indicate that constitutively active 7TM-GPCRs can nevertheless be regulated by receptor-binding ligands via a process of inverse agonism rather than by acting as traditional agonists or antagonists.
Our experiments suggest a potential role for CXCR7 in tumor development. In separate experiments, we have surveyed a broad panel of primary human tumors for CXCR7 expression by immunohistochemistry and found that many human tumors, as well as the neovasculature feeding these tumors (but not normal blood vessels), express CXCR7 (unpublished data). It is not yet clear whether the suppressed tumor growth observed in the presence of CXCR7-binding small molecules in vivo () reflects action on tumor CXCR7, vasculature CXCR7, or both. In support of a direct role for CXCR7 on the tumor cells, we have done an extensive series of RNAi experiments showing that 90% reduction of CXCR7 expression on two separate tumor lines leads to dramatic reduction of in vivo growth of these tumors (unpublished data). We have also observed that introduction of CXCR7 into MDA MB435s cells transforms their in vivo growth from exceedingly slow to dramatically faster (unpublished data). Although these data implicate the importance of tumor-associated CXCR7, they do not exclude an additional role of CXCR7 expressed by neovasculature in tumor development.
Like CXCR4 and CCR5, CXCR7/RDC1 has been shown to be a coreceptor for HIV and SIV, in this case strains that are neither M- nor T-tropic (36
). However, the role of CXCR7/RDC1 in the transmission and pathogenesis of HIV and SIV remains to be elucidated. A study published in late 2005 by Balabanian et al. (37
) evaluated the shared HIV coreceptor function of CXCR4 and RDC1 and found that SDF-1–induced T cell chemotaxis could be blocked by specific antibodies to either CXCR4 or RDC1. Balabanian's work reproduced our finding (14
) that both receptors used SDF-1 ligand; however, a major distinction between their study and our own is that SDF-1 binding to CXCR7 caused T cell chemotaxis in their hands, whereas we have not observed RDC1-mediated chemotaxis of any cell tested to date, including primary T cells (unpublished data). Furthermore, we have not detected surface CXCR7 on mouse or human T cells, either by radiolabeled SDF-1 binding analyses or CXCR7-specific mAb binding (unpublished data). The basis of the discrepant observations of Balabanian's study and our own is currently unknown and warrants further investigation.
In conclusion, CXCR7 is a new CXCR with properties that affect a spectrum of important biological and pathological processes, including cell growth/survival and adhesion, as well as the promotion of tumor growth. Our data suggest that the homeostatic and inflammatory events regulated by SDF-1 and I-TAC are much more complex than previously thought and that reinterpretation of earlier findings related to these chemokines in light of finding an additional high affinity receptor for them may be warranted. CXCR7 may provide a new molecular link in the chain of connections between inflammation and cancer, and in this context the interrelationships between CXCR7, CXCR4, CXCR3, and their shared chemokines, SDF-1 and I-TAC, will be of considerable interest. Finally, the elucidation of this new receptor may introduce new avenues of potential therapeutic intervention in important clinical indications, including oncology.