CXCR7 expression disrupts endothelial cell homeostasis and causes ligand-dependent invasion

The homeostatic function of endothelial cells (EC) is critical for a number of physiological processes including vascular integrity, immunity, and wound healing. Indeed, vascular abnormalities resulting from EC dysfunction contribute to the development and spread of malignancies. The alternative SDF-1/CXCL12 receptor CXCR7 is frequently and specifically highly expressed in tumor-associated vessels. In this study, we investigate whether CXCR7 contributes to vascular dysfunction by specifically examining the effect of CXCR7 expression on EC barrier function and motility. We demonstrate that CXCR7 expression in EC results in redistribution of CD31/PECAM-1 and loss of contact inhibition. Moreover, CXCR7+ EC are deficient in barrier formation. We show that CXCR7-mediated motility has no influence on angiogenesis but contributes to another motile process, the invasion of CXCR7+ EC into ligand-rich niches. These results identify CXCR7 as a novel manipulator of EC barrier function via alteration of PECAM-1 homophilic junctions. As such, aberrant expression of CXCR7 in the vasculature has the potential to disrupt vascular homeostasis and could contribute to vascular dysfunction in cancer systems.     

Chemokine Receptor CXCR7 Is a Functional Receptor for CXCL12 in Brain Endothelial Cells

The chemokine CXCL12 regulates multiple cell functions through its receptor, CXCR4. However, recent studies have shown that CXCL12 also binds a second receptor, CXCR7, to potentiate signal transduction and cell activity. In contrast to CXCL12/CXCR4, few studies have focused on the role of CXCR7 in vascular biology and its role in human brain microvascular endothelial cells (HBMECs) remains unclear. In this report, we used complementary methods, including immunocytofluorescence, Western blot, and flow cytometry analyses, to demonstrate that CXCR7 was expressed on HBMECs. We then employed short hairpin RNA (shRNA) technology to knockdown CXCR7 in HBMECs. Knockdown of CXCR7 in HBMECs resulted in significantly reduced HBMEC proliferation, tube formation, and migration, as well as adhesion to matrigel and tumor cells. Blocking CXCR7 with a specific antibody or small molecule antagonist similarly disrupted HBMEC binding to matrigel or tumor cells. We found that tumor necrosis factor (TNF)-α induced CXCR7 in a time and dose-response manner and that this increase preceded an increase in vascular cell adhesion molecule-1 (VCAM-1). Knockdown of CXCR7 resulted in suppression of VCAM-1, suggesting that the reduced binding of CXCR7-knockdown HBMECs may result from suppression of VCAM-1. Collectively, CXCR7 acted as a functional receptor for CXCL12 in brain endothelial cells. Targeting CXCR7 in tumor vasculature may provide novel opportunities for improving brain tumor therapy.     

趋化因子CXCL12及其受体CXCR4在胶质瘤中的研究进展

CXCL12是趋化因子家族成员之一,是能够特异性结合其受体CXCR4发挥趋化性作用的细胞因子。最初,CXCL12及CXCR4被发现于炎症细胞,参与机体炎症、免疫等病理反应。接下来的几年中发现,它在机体发育、成熟过程中也有重要作用。如今,大量研究表明它与肿瘤的生长、侵袭及转移密切相关。据报道,在乳腺癌、肺癌、卵巢癌等二十余种肿瘤组织中发现CXCL12及CXCR4的表达,其中也包括中枢系统肿瘤-胶质瘤。CXCL12/CXCR4参与胶质瘤生长过程的多个步骤,包括肿瘤增殖、侵袭、转移等。有实验指出,转移灶的CXCR4表达水平较原发灶高,CXCR4有可能成为抑制胶质瘤生长、转移的重要靶目标。     

Involvement of the CXCR7/CXCR4/CXCL12 Axis in the Malignant Progression of Human Neuroblastoma

Neuroblastoma (NB) is a typical childhood and heterogeneous neoplasm for which efficient targeted therapies for high-risk tumors are not yet identified. The chemokine CXCL12, and its receptors CXCR4 and CXCR7 have been involved in tumor progression and dissemination. While CXCR4 expression is associated to undifferentiated tumors and poor prognosis, the role of CXCR7, the recently identified second CXCL12 receptor, has not yet been elucidated in NB. In this report, CXCR7 and CXCL12 expressions were evaluated using a tissue micro-array including 156 primary and 56 metastatic NB tissues. CXCL12 was found to be highly associated to NB vascular and stromal structures. In contrast to CXCR4, CXCR7 expression was low in undifferentiated tumors, while its expression was stronger in matured tissues and specifically associated to differentiated neural tumor cells. As determined by RT-PCR, CXCR7 expression was mainly detected in N-and S-type NB cell lines, and was slightly induced upon NB cell differentiation in vitro. The relative roles of the two CXCL12 receptors were further assessed by overexpressing CXCR7 or CXCR4 receptor alone, or in combination, in the IGR-NB8 and the SH-SY5Y NB cell lines. In vitro functional analyses indicated that, in response to their common ligand, both receptors induced activation of ERK1/2 cascade, but not Akt pathway. CXCR7 strongly reduced in vitro growth, in contrast to CXCR4, and impaired CXCR4/CXCL12-mediated chemotaxis. Subcutaneous implantation of CXCR7-expressing NB cells showed that CXCR7 also significantly reduced in vivo growth. Moreover, CXCR7 affected CXCR4-mediated orthotopic growth in a CXCL12-producing environment. In such model, CXCR7, in association with CXCR4, did not induce NB cell metastatic dissemination. In conclusion, the CXCR7 and CXCR4 receptors revealed specific expression patterns and distinct functional roles in NB. Our data suggest that CXCR7 elicits anti-tumorigenic functions, and may act as a regulator of CXCR4/CXCL12-mediated signaling in NB.     

Chemokine receptor trio: CXCR3, CXCR4 and CXCR7 crosstalk via CXCL11 and CXCL12

Although chemokines are well established to function in immunity and endothelial cell activation and proliferation, a rapidly growing literature suggests that CXC Chemokine receptors CXCR3, CXCR4 and CXCR7 are critical in the development and progression of solid tumors. The effect of these chemokine receptors in tumorigenesis is mediated via interactions with shared ligands I-TAC (CXCL11) and SDF-1 (CXCL12). Over the last decade, CXCR4 has been extensively reported to be overexpressed in most human solid tumors and has earned considerable attention toward elucidating its role in cancer metastasis. To enrich the existing armamentarium of anti-cancerous agents, many inhibitors of CXCL12–CXCR4 axis have emerged as additional or alternative agents for neo-adjuvant treatments and even many of them are in preclinical and clinical stages of their development. However, the discovery of CXCR7 as another receptor for CXCL12 with rather high binding affinity and recent reports about its involvement in cancer progression, has questioned the potential of “selective blockade” of CXCR4 as cancer chemotherapeutics. Interestingly, CXCR7 can also bind another chemokine CXCL11, which is an established ligand for CXCR3. Recent reports have documented that CXCR3 and their ligands are overexpressed in different solid tumors and regulate tumor growth and metastasis. Therefore, it is important to consider the interactions and crosstalk between these three chemokine receptors and their ligand mediated signaling cascades for the development of effective anti-cancer therapies. Emerging evidence also indicates that these receptors are differentially expressed in tumor endothelial cells as well as in cancer stem cells, suggesting their direct role in regulating tumor angiogenesis and metastasis. In this review, we will focus on the signals mediated by this receptor trio via their shared ligands and their role in tumor growth and progression.     

The role of CXCR7/RDC1 as a chemokine receptor for CXCL12/SDF-1 in prostate cancer

Several reports have recently documented that CXCR7/RDC1 functions as a chemokine receptor for SDF-1/CXCL12, which regulates a spectrum of normal and pathological processes. In this study, the role of CXCR7/RDC1 in prostate cancer (PCa) was explored. Staining of high density tissue microarrays demonstrates that the levels of CXCR7/RDC1 expression increase as the tumors become more aggressive. In vitro and in vivo studies with PCa cell lines suggest that alterations in CXCR7/RDC1 expression are associated with enhanced adhesive and invasive activities in addition to a survival advantage. In addition, it was observed that CXCR7/RDC1 levels are regulated by CXCR4. Among the potential downstream targets of CXCR7/RDC1 are CD44 and cadherin-11, which are likely to contribute to the invasiveness of PCa cells. CXCR7/RDC1 also regulates the expression of the proangiogenic factors interleukin-8 or vascular endothelial growth factor, which are likely to participate in the regulation of tumor angiogenesis. Finally, we found that signaling by CXCR7/RDC1 activates AKT pathways. Together, these data demonstrate a role for CXCR7/RDC1 in PCa metastasis and progression and suggest potential targets for therapeutic intervention.     

Early postnatal lethality and cardiovascular defects in CXCR7-deficient mice

CXCR7 is a G-protein coupled receptor that was recently deorphanized and shown to have SDF1 and I-TAC as high affinity ligands. Here we describe the characterization of CXCR7-deficient mice that were generated to further investigate the function of this receptor in vivo. Expression analysis using a LacZ reporter knockin revealed that postnatally Cxcr7 was specifically expressed in cardiomyocytes, vascular endothelial cells of the lung and heart, the cerebral cortex and in osteocytes of the bone. Adult tissues revealed high expression in cardiomyocytes and osteocytes. The observation that 70% of the Cxcr7-/- mice died in the first week after birth coincides with expression of Cxcr7 in vascular endothelial cells and in cardiomyocytes. An important role of CXCR7 in the cardiovascular system was further supported by the observation that hearts of the Cxcr7-/- mice were enlarged, showed myocardial degeneration and fibrosis of postnatal origin, and hyperplasia of embryonic origin. Despite high expression in osteocytes no apparent bone phenotype was observed, neither in combination with ovariectomy nor orchidectomy. Thus as CXCR7 does not seem to play an important role in bone our data indicate an important function of CXCR7 in the cardiovascular system during multiple steps of development.     

Stromal cell-derived factor-1alpha (SDF-1alpha/CXCL12) stimulates ovarian cancer cell growth through the EGF receptor transactivation

Ovarian cancer (OC) is the leading cause of death in gynecologic diseases in which there is evidence for a complex chemokine network. Chemokines are a family of proteins that play an important role in tumor progression influencing cell proliferation, angiogenic/angiostatic processes, cell migration and metastasis, and, finally, regulating the immune cells recruitment into the tumor mass. We previously demonstrated that astrocytes and glioblastoma cells express both the chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1), and that SDF-1alpha treatment induced cell proliferation, supporting the hypothesis that chemokines may play an important role in tumor cells' growth in vitro. In the present study, we report that CXCR4 and SDF-1 are expressed in OC cell lines. We demonstrate that SDF-1alpha induces a dose-dependent proliferation in OC cells, by the specific interaction with CXCR4 and a biphasic activation of ERK1/2 and Akt kinases. Our results further indicate that CXCR4 activation induces EGF receptor (EGFR) phosphorylation that in turn was linked to the downstream intracellular kinases activation, ERK1/2 and Akt. In addition, we provide evidence for cytoplasmic tyrosine kinase (c-Src) involvement in the SDF-1/CXCR4-EGFR transactivation. These results suggest a possible important "cross-talk" between SDF-1/CXCR4 and EGFR intracellular pathways that may link signals of cell proliferation in ovarian cancer.     

CXCL12 mediates trophic interactions between endothelial and tumor cells in glioblastoma

Emerging evidence suggests endothelial cells (EC) play a critical role in promoting Glioblastoma multiforme (GBM) cell proliferation and resistance to therapy. The molecular basis for GBM-EC interactions is incompletely understood. We hypothesized that the chemokine CXCL12 and its receptor CXCR4 could mediate direct interactions between GBM cells and tumor-associated endothelial cells and that disruption of this interaction might be the molecular basis for the anti-tumor effects of CXCR4 antagonists. We investigated this possibility in vivo and in an in vitro co-culture model that incorporated extracellular matrix, primary human brain microvascular ECs (HBMECs) and either an established GBM cell line or primary GBM specimens. Depletion of CXCR4 in U87 GBM cells blocked their growth as intracranial xenografts indicating that tumor cell CXCR4 is required for tumor growth in vivo. In vitro, co-culture of either U87 cells or primary GBM cells with HBMECs resulted in their co-localization and enhanced GBM cell growth. Genetic manipulation of CXCL12 expression and pharmacological inhibition of its receptors CXCR4 and CXCR7 revealed that the localizing and trophic effects of endothelial cells on GBM cells were dependent upon CXCL12 and CXCR4. These findings indicate that the CXCL12/CXCR4 pathway directly mediates endothelial cell trophic function in GBMs and that inhibition of CXCL12-CXCR4 signaling may uniquely target this activity. Therapeutic disruption of endothelial cell trophic functions could complement the structural disruption of anti-angiogenic regimens and, in combination, might also improve the efficacy of radiation and chemotherapy in treating GBMs.     

CXCR3, a double-edged sword in tumor progression and angiogenesis

CXC chemokines are involved in chemotaxis, regulation of cell growth, induction of apoptosis and modulation of angiostatic effects. CXCL9, CXCL10, CXCL11, CXCL4 and its variant CXCL4L1 are members of the CXC chemokine family, which bind to the CXCR3 receptor to exert their biological effects. These chemokines are associated with a variety of human diseases including chronic inflammation, immune dysfunction, cancer and metastasis. In this review, we focus on accumulating evidence demonstrating the pivotal role of CXCR3 in tumor progression. Its effects are mediated directly in tumor cells or indirectly through the regulation of angiogenesis and tumor immunity. Understanding the emerging role of CXCR3 and its signaling mechanisms further validates this receptor as a biomarker and therapeutic target for tumor progression and tumor angiogenesis.     

CXCR2-Dependent Endothelial Progenitor Cell Mobilization in Pancreatic Cancer Growth

Neovascularization is essential for tumor growth. We have previously reported that the chemokine receptor CXCR2 is an important regulator in tumor angiogenesis. Here we report that the mobilization of bone marrow (BM)-derived endothelial progenitor cells (EPCs) is impaired in CXCR2 knockout mice harboring pancreatic cancers. The circulating levels of EPCs (positive for CD34, CD117, CD133, or CD146) are decreased in the bone marrow and/or blood of tumor-bearing CXCR2 knockout mice. CXCR2 gene knockout reduced BM-derived EPC proliferation, differentiation, and vasculogenesis in vitro. EPCs double positive for CD34 and CD133 increased tumor angiogenesis and pancreatic cancer growth in vivo. In addition, CD133(+) and CD146(+) EPCs in human pancreatic cancer are increased compared with normal pancreas tissue. These findings indicate a role of BM-derived EPC in pancreatic cancer growth and provide a cellular mechanism for CXCR2 mediated tumor neovascularization.     

A point mutation (G574A) in the chemokine receptor CXCR4 detected in human cancer cells enhances migration

The chemokine receptor CXCR4 is widely expressed in human cancers and regulates cell invasion, proliferation and survival. Because mutations in the CXCR4 gene could regulate its function we sequenced the coding region of the CXCR4 gene in 18 human melanoma and 3 human colon carcinoma cell lines. The same somatic point mutation (G574A; V160I) in the fourth trans-membrane region of CXCR4 was detected in one colon cancer cell line (PD) and one melanoma cell line (LB). CXCR4 was expressed and functional in both PD and LB cells, PD and LB cells migrated specifically toward the receptor ligand, CXCL12 and P-Erk was specifically induced by CXCL12. To give insight into the function of the mutant CXCR4 receptor, human A431, epidermoid carcinoma cells, were stably transfected with both mutant and wild type CXCR4. In vitro, A431 cells harboring CXCR4^G574A migrated specifically toward CXCL12 and CXCL12 induced ERK phosphorylation. Interestingly, in vivo studies showed that the growth of A431 tumors harboring CXCR4G574A was delayed compared to those harboring WT CXCR4. As expected, treatment with AMD3100, a specific CXCR4 inhibitor, reduced the in vivo growth of CXCR4_^G574A tumor b^G574A rprisingly, increased the growth of CXCR4^G574A A431 cells. This is the first report of a spontaneously occurring, functionally active CXCR4 mutation in human cancer cells. While the mutation impairs cell growth in vivo, the CXCR4 inhibitor, AMD3100, stimulated the growth of cells harboring CXCR4^G574A.     

Hepatocyte growth factor enhances CXCR4 expression favoring breast cancer cell invasiveness

Microenvironmental factors affect different aspects of tumor cell biology, including cell survival, invasion, and metastasis. Here, we report that hepatocyte growth factor and hypoxia may contribute to breast carcinoma cell invasiveness by inducing the chemokine receptor CXCR4. Hepatocyte growth factor enhanced CXCR4 mRNA and protein expression exclusively in MCF-7 (low invasive) carcinoma cells, while in response to hypoxia, CXCR4 induction was observed in both MCF-7 and MDA-MB 231 (highly invasive) carcinoma cells. The receptor induction had a functional role in cancer cells, as demonstrated by the fact that hepatocyte growth factor pretreatment promoted MCF-7 cell migration toward the CXCR4-specific ligand CXCL12. Extracellular signal-regulated protein kinase 1/2 (ERK1/2) and phosphoinositide-3-kinase (PI3K) transduction pathways seemed to be differently implicated in the early induction of CXCR4 by hepatocyte growth factor or hypoxia in the two breast carcinoma cells examined.     

Llama-derived Single Variable Domains (Nanobodies) Directed against Chemokine Receptor CXCR7 Reduce Head and Neck Cancer Cell Growth in Vivo

The chemokine receptor CXCR7, belonging to the membrane-bound G protein-coupled receptor superfamily, is expressed in several tumor types. Inhibition of CXCR7 with either small molecules or small interference (si)RNA has shown promising therapeutic benefits in several tumor models. With the increased interest and effectiveness of biologicals inhibiting membrane-bound receptors we made use of the “Nanobody platform” to target CXCR7. Previously we showed that Nanobodies, i.e. immunoglobulin single variable domains derived from naturally occurring heavy chain-only camelids antibodies, represent new biological tools to efficiently tackle difficult drug targets such as G protein-coupled receptors. In this study we developed and characterized highly selective and potent Nanobodies against CXCR7. Interestingly, the CXCR7-targeting Nanobodies displayed antagonistic properties in contrast with previously reported CXCR7-targeting agents. Several high affinity CXCR7-specific Nanobodies potently inhibited CXCL12-induced β-arrestin2 recruitment in vitro. A wide variety of tumor biopsies was profiled, showing for the first time high expression of CXCR7 in head and neck cancer. Using a patient-derived CXCR7-expressing head and neck cancer xenograft model in nude mice, tumor growth was inhibited by CXCR7-targeting Nanobody therapy. Mechanistically, CXCR7-targeting Nanobodies did not inhibit cell cycle progression but instead reduced secretion of the angiogenic chemokine CXCL1 from head and neck cancer cells in vitro, thus acting here as inverse agonists, and subsequent angiogenesis in vivo. Hence, with this novel class of CXCR7 inhibitors, we further substantiate the therapeutic relevance of targeting CXCR7 in head and neck cancer.     

Activation of CXCR7 promotes endothelial repair and reduces the carotid atherosclerotic lesions through inhibition of pyroptosis signaling pathways

Endothelial injuries, including cell pyroptosis, are ongoing inflammatory processes with key roles in atherosclerosis development. Our previous report showed that the chemokine CXCL12 and its receptor CXCR7 are associated with the proliferation and angiogenesis of endothelial cells. Nevertheless, the mechanism underlying these effects on atherosclerotic lesions, especially on endothelial dysfunction, remains unknown. Here, we demonstrated that CXCR7 was upregulated in human carotid atherosclerotic plaques, apolipoprotein E knockout (ApoE^?/?) mice fed with a high‐fat diet (HFD), and oxidized lipopolysaccharide‐treated (ox‐LDL) human umbilical vein endothelial cells (HUVECs). Further, the activation of CXCR7 reversed ox‐LDL‐induced HUVEC dysfunction, such as migration, tube formation, and cell pyroptosis; all of these protective effects were alleviated by inhibition of CXCR7. The NOD‐like receptor family pyrin domain‐containing 3 (NLRP3) inflammasomes were also elevated in human carotid atherosclerotic plaques, ApoE^?/? mice fed with HFD, and ox‐LDL‐injured HUVECs by regulation of caspase‐1 and interleukin (IL)‐1β expression. The activation of CXCR7 by TC14012 led to a decrease in atherosclerotic lesions in ApoE^?/? mice fed with HFD. TC14012 also inhibited the expression of the NLRP3 inflammasome signaling pathway in vivo. In conclusion, our study suggests that CXCR7 plays an important role in regulating NLRP3 inflammasome‐modulated pyroptosis in HUVECs, providing a potential novel therapy for atherosclerosis.     

Crosstalk between chemokine receptor CXCR4 and cannabinoid receptor CB2 in modulating breast cancer growth and invasion

Background

Cannabinoids bind to cannabinoid receptors CB₁ and CB₂ and have been reported to possess anti-tumorigenic activity in various cancers. However, the mechanisms through which cannabinoids modulate tumor growth are not well known. In this study, we report that a synthetic non-psychoactive cannabinoid that specifically binds to cannabinoid receptor CB₂ may modulate breast tumor growth and metastasis by inhibiting signaling of the chemokine receptor CXCR4 and its ligand CXCL12. This signaling pathway has been shown to play an important role in regulating breast cancer progression and metastasis.

Methodology/Principal Findings

We observed high expression of both CB₂ and CXCR4 receptors in breast cancer patient tissues by immunohistochemical analysis. We further found that CB₂-specific agonist JWH-015 inhibits the CXCL12-induced chemotaxis and wound healing of MCF7 overexpressing CXCR4 (MCF7/CXCR4), highly metastatic clone of MDA-MB-231 (SCP2) and NT 2.5 cells (derived from MMTV-neu) by using chemotactic and wound healing assays. Elucidation of the molecular mechanisms using various biochemical techniques and confocal microscopy revealed that JWH-015 treatment inhibited CXCL12-induced P44/P42 ERK activation, cytoskeletal focal adhesion and stress fiber formation, which play a critical role in breast cancer invasion and metastasis. In addition, we have shown that JWH-015 significantly inhibits orthotopic tumor growth in syngenic mice in vivo using NT 2.5 cells. Furthermore, our studies have revealed that JWH-015 significantly inhibits phosphorylation of CXCR4 and its downstream signaling in vivo in orthotopic and spontaneous breast cancer MMTV-PyMT mouse model systems.

Conclusions/Significance

This study provides novel insights into the crosstalk between CB₂ and CXCR4/CXCL12-signaling pathways in the modulation of breast tumor growth and metastasis. Furthermore, these studies indicate that CB₂ receptors could be used for developing innovative therapeutic strategies against breast cancer.     

Ubiquitination of CXCR7 controls receptor trafficking

The chemokine receptor CXCR7 binds CXCL11 and CXCL12 with high affinity, chemokines that were previously thought to bind exclusively to CXCR4 and CXCR3, respectively. Expression of CXCR7 has been associated with cardiac development as well as with tumor growth and progression. Despite having all the canonical features of G protein-coupled receptors (GPCRs), the signalling pathways following CXCR7 activation remain controversial, since unlike typical chemokine receptors, CXCR7 fails to activate Gα(i)-proteins. CXCR7 has recently been shown to interact with β-arrestins and such interaction has been suggested to be responsible for G protein-independent signals through ERK-1/2 phosphorylation. Signal transduction by CXCR7 is controlled at the membrane by the process of GPCR trafficking. In the present study we investigated the regulatory processes triggered by CXCR7 activation as well as the molecular interactions that participate in such processes. We show that, CXCR7 internalizes and recycles back to the cell surface after agonist exposure, and that internalization is not only β-arrestin-mediated but also dependent on the Serine/Threonine residues at the C-terminus of the receptor. Furthermore we describe, for the first time, the constitutive ubiquitination of CXCR7. Such ubiquitination is a key modification responsible for the correct trafficking of CXCR7 from and to the plasma membrane. Moreover, we found that CXCR7 is reversibly de-ubiquitinated upon treatment with CXCL12. Finally, we have also identified the Lysine residues at the C-terminus of CXCR7 to be essential for receptor cell surface delivery. Together these data demonstrate the differential regulation of CXCR7 compared to the related CXCR3 and CXCR4 receptors, and highlight the importance of understanding the molecular determinants responsible for this process.     

The peptidomimetic CXCR4 antagonist TC14012 recruits beta-arrestin to CXCR7: roles of receptor domains

CXCR7 is an atypical chemokine receptor that signals through β-arrestin in response to agonists without detectable activation of heterotrimeric G-proteins. Its cognate chemokine ligand CXCL12 also binds CXCR4, a chemokine receptor of considerable clinical interest. Here we report that TC14012, a peptidomimetic inverse agonist of CXCR4, is an agonist on CXCR7. The potency of β-arrestin recruitment to CXCR7 by TC14012 is much higher than that of the previously reported CXCR4 antagonist AMD3100 and differs only by one log from that of the natural ligand CXCL12 (EC(50) 350 nM for TC14012, as compared with 30 nM for CXCL12 and 140 μM for AMD3100). Moreover, like CXCL12, TC14012 leads to Erk 1/2 activation in U373 glioma cells that express only CXCR7, but not CXCR4. Given that with TC14012 and AMD3100 two structurally unrelated CXCR4 antagonists turn out to be agonists on CXCR7, this likely reflects differences in the activation mechanism of the arrestin pathway by both receptors. To identify the receptor domain responsible for these opposed effects, we investigated CXCR4 and CXCR7 C terminus-swapping chimeras. Using quantitative bioluminescence resonance energy transfer, we find that the CXCR7 receptor core formed by the seven-transmembrane domains and the connecting loops determines the agonistic activity of both TC14012 and AMD3100. Moreover, we find that the CXCR7 chimera bearing the CXCR4 C-terminal constitutively associates with arrestin in the absence of ligands. Our data suggest that the CXCR4 and CXCR7 cores share ligand-binding surfaces for the binding of the synthetic ligands, indicating that CXCR4 inhibitors should be tested also on CXCR7.     

Third Extracellular Loop (EC3)-N Terminus Interaction Is Important for Seven-transmembrane Domain Receptor Function: IMPLICATIONS FOR AN ACTIVATION MICROSWITCH REGION*

The canonical heptahelical bundle architecture of seven-transmembrane domain (7TM) receptors is intertwined by three intra- and three extracellular loops, whose local conformations are important in receptor signaling. Many 7TM receptors contain a cysteine residue in the third extracellular loop (EC3) and a complementary cysteine residue on the N terminus. The functional role of such EC3-N terminus conserved cysteine pairs remains unclear. This study explores the role of the EC3-N terminus cysteine pairs on receptor conformation and G protein activation by disrupting them in the chemokine receptor CXCR4, while engineering a novel EC3-N terminus cysteine pair into the complement factor 5a receptor (C5aR), a chemo attractant receptor that lacks it. Mutated CXCR4 and C5aRs were expressed in engineered yeast. Mutation of the cysteine pair with the serine pair (C28S/C274S) in constitutively active mutant CXCR4 abrogated the receptor activation, whereas mutation with the aromatic pair (C28F–C274F) or the salt bridge pair (C28R/C274E), respectively, rescued or retained the receptor activation in response to CXCL12. In this context, the cysteine pair (Cys³⁰ and Cys²⁷²) engineered into the EC3-N terminus (Ser³⁰ and Ser²⁷²) of a novel constitutively active mutant of C5aR restrained the constitutive signaling without affecting the C5a-induced activation. Further mutational studies demonstrated a previously unappreciated role for Ser²⁷² on EC3 of C5aR and its interaction with the N terminus, thus defining a new microswitch region within the C5aR. Similar results were obtained with mutated CXCR4 and C5aRs expressed in COS-7 cells. These studies demonstrate a novel role of the EC3-N terminus cysteine pairs in G protein-coupled receptor activation and signaling.