G-protein-dependent and -independent pathways in denatonium signal transduction

To clarify the involvement of G protein in denatonium signal transduction, we carried out a whole-cell patch-clamp analysis with isolated taste cells in mice. Two different responses were observed by applying GDP-beta-S, a G-protein inhibitor. One response to denatonium was reduced by GDP-beta-S (G-protein-dependent), whereas the other was not affected (G-protein-independent). These different patterns were also observed by concurrently inhibiting the phospholipase C beta2 and phosphodiesterase pathways via G protein. These data suggest dual, G-protein-dependent and -independent mechanisms for denatonium. Moreover, the denatonium responses were not attenuated by singly inhibiting the phospholipase C beta2 or phosphodiesterase pathway, implying that both pathways were involved in G-protein-dependent transduction. In the G-protein-independent cells, the response was abolished by the depletion of calcium ions within the intracellular store. These results suggest that Ca2+ release from the intracellular store is an important factor. Our data demonstrate multiple transduction pathways for denatonium in mammalian taste cells.     

Label-free impedance responses of endogenous and synthetic chemokine receptor CXCR3 agonists correlate with Gi-protein pathway activation

The chemokine receptor CXCR3 is a G-protein-coupled receptor that signals through the Gα(i) class of heterotrimeric G-proteins. CXCR3 is highly expressed on activated T cells and has been proposed to be a therapeutic target in autoimmune disease. CXCR3 is activated by the chemokines CXCL9, CXCL10 and CXCL11. CXCR3 signaling properties in response to CXCL10, CXCL11 and the synthetic agonist VUF10661 have previously been evaluated using conventional endpoint assays. In the present study, label-free impedance measurements were used to characterize holistic responses of CXCR3-expressing cells to stimulation with chemokines and VUF10661 in real time and to compare these responses with both G-protein and non-G-protein (β-arrestin2) mediated responses. Differences in response kinetics were apparent between the chemokines and VUF10661. Moreover, CXCR3-independent effects could be distinguished from CXCR3-specific responses with the use of the selective CXCR3 antagonist NBI-74330 and the Gα(i) inhibitor pertussis toxin. By comparing the various responses, we observed that CXCL9 is a biased CXCR3 agonist, stimulating solely G-protein-dependent pathways. Moreover, CXCR3-mediated changes in cellular impedance correlated with G-protein signaling, but not β-arrestin2 recruitment.     

RGS4 inhibits platelet-activating factor receptor phosphorylation and cellular responses

To define the role of regulators of G-protein signaling (RGS) in chemoattractant-mediated responses, RGS4 and the receptors for platelet-activating factor (PAFR), formylated peptides (FR), or interleukin-8 (CXCR1) were stably coexpressed in a rat basophilic leukemia (RBL-2H3) cell line. The data demonstrate that RGS4 inhibited responses by PAFR (i.e., phosphoinositide (PI) hydrolysis, Ca2+ mobilization) but not by FR or CXCR1. An N-terminal 33 amino acid deletion mutant of RGS4 (DeltaRGS4), deficient in GAP (GTPase activating protein) activity and plasma membrane localization, had no effect on either PAFR, FR, or CXCR1. RGS4, but not DeltaRGS4, also blocked phosphorylation of PAFR by platelet-activating factor (PAF) and, unexpectedly, by phorbol 12-myristate 13-acetate (PMA); it also blocked cross-phosphorylation by formylmethionylleucylphenylalanine (fMLP). A point mutant of RGS4 (N88S), deficient in GAP activity but not membrane localization, partially blocked PAFR phosphorylation but had no effect on PAFR-mediated PI hydrolysis and Ca2+ mobilization. Truncation of the cytoplasmic tail of PAFR (mPAFR) resulted in a loss of its susceptibility to inhibition by RGS4. Taken together, the data indicate that of the receptors studied, RGS4 selectively inhibited responses to PAFR, which preferentially couples to Gq. At the level of expression studied, RGS4 did not inhibit FR or CXCR1 which activates Gi to transduce cellular signals. Since the tail-deleted mutant of PAFR was not affected by RGS4, and RGS4 blocked homologous as well as heterologous phosphorylation of this receptor, it is possible that RGS4 interferes sterically with the cytoplasmic tail of PAFR. Thus, in addition to stimulating the GTPase activity of Galpha, RGS4 prevents G protein activation by PAFR and the homologous and heterologous phosphorylation of this receptor.     

CXCL12 and [N33A]CXCL12 in 5637 and HeLa cells: regulating HER1 phosphorylation via calmodulin/calcineurin

In the human neoplastic cell lines 5637 and HeLa, recombinant CXCL12 elicited, as expected, downstream signals via both G-protein-dependent and β-arrestin-dependent pathways responsible for inducing a rapid and a late wave, respectively, of ERK1/2 phosphorylation. In contrast, the structural variant [N33A]CXCL12 triggered no β-arrestin-dependent phosphorylation of ERK1/2, and signaled via G protein-dependent pathways alone. Both CXCL12 and [N33A]CXCL12, however, generated signals that transinhibited HER1 phosphorylation via intracellular pathways. 1) Prestimulation of CXCR4/HER1-positive 5637 or HeLa cells with CXCL12 modified the HB-EGF-dependent activation of HER1 by delaying the peak phosphorylation of tyrosine 1068 or 1173. 2) Prestimulation with the synthetic variant [N33A]CXCL12, while preserving CXCR4-related chemotaxis and CXCR4 internalization, abolished HER1 phosphorylation. 3) In cells knockdown of β-arrestin 2, CXCL12 induced a full inhibition of HER1 like [N33A]CXCL12 in non-silenced cells. 4) HER1 phosphorylation was restored as usual by inhibiting PCK, calmodulin or calcineurin, whereas the inhibition of CaMKII had no discernable effect. We conclude that both recombinant CXCL12 and its structural variant [N33A]CXCL12 may transinhibit HER1 via G-proteins/calmodulin/calcineurin, but [N33A]CXCL12 does not activate β-arrestin-dependent ERK1/2 phosphorylation and retains a stronger inhibitory effect. Therefore, we demonstrated that CXCL12 may influence the magnitude and the persistence of signaling downstream of HER1 in turn involved in the proliferative potential of numerous epithelial cancer. In addition, we recognized that [N33A]CXCL12 activates preferentially G-protein-dependent pathways and is an inhibitor of HER1.     

Structural requirements for CD43 function

The regulation of T cell activation and adhesion by CD43 (leukosialin, sialophorin) has been thought to be mainly a function of the large size and negative charge of the extracellular domain of the protein. In this work, we demonstrate that the cytoplasmic tail is both necessary and sufficient for the negative regulatory effect of CD43 on cell-cell adhesion. Expression of mutant CD43 proteins in primary T cells from CD43-deficient mice demonstrated that the antiproliferative effect of CD43 is also dependent upon the cytoplasmic tail. In contrast, Ab-mediated costimulation through CD43 does not require the intracellular domain of CD43. These data demonstrate that CD43 primarily serves as a negative regulator of T cell activation and adhesion, and that this is mediated not exclusively by passive effects of the extracellular domain, but requires participation of the cytoplasmic tail, perhaps through interactions with the cytoskeleton, or alternatively, active regulation of intracellular signaling pathways.     

How do chemokines navigate neutrophils to the target site: Dissecting the structural mechanisms and signaling pathways

Chemokines play crucial roles in combating microbial infection and initiating tissue repair by recruiting neutrophils in a timely and coordinated manner. In humans, no less than seven chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8) and two receptors (CXCR1 and CXCR2) mediate neutrophil functions but in a context dependent manner. Neutrophil-activating chemokines reversibly exist as monomers and dimers, and their receptor binding triggers conformational changes that are coupled to G-protein and β-arrestin signaling pathways. G-protein signaling activates a variety of effectors including Ca²⁺ channels and phospholipase C. β-arrestin serves as a multifunctional adaptor and is coupled to several signaling hubs including MAP kinase and tyrosine kinase pathways. Both G-protein and β-arrestin signaling pathways play important non-overlapping roles in neutrophil trafficking and activation. Functional studies have established many similarities but distinct differences for a given chemokine and between chemokines at the level of monomer vs. dimer, CXCR1 vs. CXCR2 activation, and G-protein vs. β-arrestin pathways. We propose that two forms of the ligand binding two receptors and activating two signaling pathways enables fine-tuned neutrophil function compared to a single form, a single receptor, or a single pathway. We summarize the current knowledge on the molecular mechanisms by which chemokine monomers/dimers activate CXCR1/CXCR2 and how these interactions trigger G-protein/β-arrestin-coupled signaling pathways. We also discuss current challenges and knowledge gaps, and likely advances in the near future that will lead to a better understanding of the relationship between the chemokine-CXCR1/CXCR2-G-protein/β-arrestin axis and neutrophil function.     

Cysteine (C)-X-C Receptor 4 Undergoes Transportin 1-Dependent Nuclear Localization and Remains Functional at the Nucleus of Metastatic Prostate Cancer Cells

The G-protein coupled receptor (GPCR), Cysteine (C)-X-C Receptor 4 (CXCR4), plays an important role in prostate cancer metastasis. CXCR4 is generally regarded as a plasma membrane receptor where it transmits signals that support transformation, progression and eventual metastasis. Due to the central role of CXCR4 in tumorigenesis, therapeutics approaches such as antagonist and monoclonal antibodies have focused on receptors that exist on the plasma membrane. An emerging concept for G-protein coupled receptors is that they may localize to and associate with the nucleus where they retain function and mediate nuclear signaling. Herein, we demonstrate that CXCR4 associated with the nucleus of malignant prostate cancer tissues. Likewise, expression of CXCR4 was detected in nuclear fractions among several prostate cancer cell lines, compared to normal prostate epithelial cells. Our studies identified a nuclear pool of CXCR4 and we defined a nuclear transport pathway for CXCR4. We reveal a putative nuclear localization sequence (NLS), ‘RPRK’, within CXCR4 that contributed to nuclear localization. Additionally, nuclear CXCR4 interacted with Transportinβ1 and Transportinβ1-binding to CXCR4 promoted its nuclear translocation. Importantly, G_αi immunoprecipitation and calcium mobilization studies indicated that nuclear CXCR4 was functional and participated in G-protein signaling, revealing that the nuclear pool of CXCR4 retained function. Given the suggestion that functional, nuclear CXCR4 may be a mechanism underlying prostate cancer recurrence, increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4, these studies addresses a novel mechanism of nuclear signaling for CXCR4, a novel mechanism of clinical targeting, and demonstrate an active nuclear pool that provides important new information to illuminate what has been primarily clinical reports of nuclear CXCR4.     

Ligand-induced partitioning of human CXCR1 chemokine receptors with lipid raft microenvironments facilitates G-protein-dependent signaling

Ligand binding to a chemokine receptor triggers signaling events through heterotrimeric G-proteins. The mechanisms underlying receptor-mediated G-protein activation in the heterogeneous microenvironments of the plasma membrane are unclear. Here, using live-cell fluorescence resonance energy transfer imaging to detect the proximity between CXCR1-cyan fluorescent protein (CFP) and fluorescence probes that label lipid raft or non-lipid raft microdomains and using fluorescence recovery after photobleaching analysis to measure the lateral diffusion of CXCR1-CFP, we found that interleukin-8 induces association between the receptors and lipid raft microenvironments. Disruption of lipid rafts impaired G-protein-dependent signaling, such as Ca2+ responses and phosphatidylinositol 3-kinase activation, but had no effect on ligand-binding function and did not completely abolish ligand-induced receptor phosphorylation. Our results suggest a novel mechanism by which ligand binding to CXCR1 promotes lipid raft partitioning of receptors and facilitates activation of heterotrimeric G-proteins.     

CXCR3 signaling reduces the severity of experimental autoimmune encephalomyelitis by controlling the parenchymal distribution of effector and regulatory T cells in the central nervous system

The chemokine receptor CXCR3 promotes the trafficking of activated T and NK cells in response to three ligands, CXCL9, CXCL10, and CXCL11. Although these chemokines are produced in the CNS in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), their role in the pathogenesis of CNS autoimmunity is unresolved. We examined the function of CXCR3 signaling in EAE using mice that were deficient for CXCR3 (CXCR3(-/-)). The time to onset and peak disease severity were similar for CXCR3(-/-) and wild-type (WT) animals; however, CXCR3(-/-) mice had more severe chronic disease with increased demyelination and axonal damage. The inflammatory lesions in WT mice consisted of well-demarcated perivascular mononuclear cell infiltrates, mainly in the spinal cord and cerebellum. In CXCR3(-/-) mice, these lesions were more widespread throughout the CNS and were diffused and poorly organized, with T cells and highly activated microglia/macrophages scattered throughout the white matter. Although the number of CD4(+) and CD8(+) T cells infiltrating the CNS were similar in CXCR3(-/-) and WT mice, Foxp3(+) regulatory T cells were significantly reduced in number and dispersed in CXCR3(-/-) mice. The expression of various chemokine and cytokine genes in the CNS was similar in CXCR3(-/-) and WT mice. The genes for the CXCR3 ligands were expressed predominantly in and/or immediately surrounding the mononuclear cell infiltrates. We conclude that in EAE, CXCR3 signaling constrains T cells to the perivascular space in the CNS and augments regulatory T cell recruitment and effector T cell interaction, thus limiting autoimmune-mediated tissue damage.     

Identification of CXCR4 domains that support coreceptor and chemokine receptor functions

The interaction of the chemokine stromal cell-derived factor 1 (SDF-1) with its receptor CXCR4 is vital for cell trafficking during development, is capable of inhibiting human immunodeficiency virus type 1 (HIV-1) utilization of CXCR4 as a coreceptor, and has been implicated in delaying disease progression to AIDS in vivo. Because of the importance of this chemokine-chemokine receptor pair to both development and disease, we investigated the molecular basis of the interaction between CXCR4 and its ligands SDF-1 and HIV-1 envelope. Using CXCR4 chimeras and mutants, we determined that SDF-1 requires the CXCR4 amino terminus for binding and activates downstream signaling pathways by interacting with the second extracellular loop of CXCR4. SDF-1-mediated activation of CXCR4 required the Asp-Arg-Tyr motif in the second intracellular loop of CXCR4, was pertussis toxin sensitive, and did not require the distal C-terminal tail of CXCR4. Several CXCR4 mutants that were not capable of binding SDF-1 or signaling still supported HIV-1 infection, indicating that the ability of CXCR4 to function as a coreceptor is independent of its ability to signal. Direct binding studies using the X4 gp120s HXB, BH8, and MN demonstrated the ability of HIV-1 gp120 to bind directly and specifically to the chemokine receptor CXCR4 in a CD4-dependent manner, using a conformationally complex structure on CXCR4. Several CXCR4 variants that did not support binding of soluble gp120 could still function as viral coreceptors, indicating that detectable binding of monomeric gp120 is not always predictive of coreceptor function.     

The CXCR1 tail mediates beta1 integrin-dependent cell migration via MAP kinase signaling

In this study, we examined how IL-8 induces leukocyte migration on major beta1 integrin ligands derived from the extracellular matrix protein fibronectin. We assessed individual contributions of signaling by IL-8 receptors by transfection of CXCR1 and CXCR2 into rat basophilic leukemia (RBL) cells and human monocytic THP-1 cells. CXCR1 expressing cells migrated on the fibronectin ligands for alpha4beta1 and alpha5beta1 integrins in response to IL-8, whereas CXCR2 expressing cells did not. RBL cells expressing the chimeric CXCR1 receptor containing the cytoplasmic tail of CXCR2 had greatly blunted migration, while cells expressing the CXCR2 chimera with the tail of CXCR1 had augmented migration. Last, inhibitors of p38 and JNK MAP kinases blocked IL-8-induced migration in CXCR1+ cells. We conclude that IL-8 stimulated beta1 integrin-mediated leukocyte migration on fibronectin through CXCR1 is dependent on the C-terminal cytoplasmic domain of CXCR1 and subsequent p38 and JNK MAPK signaling.     

Cannabinoid receptor 2-mediated attenuation of CXCR4-tropic HIV infection in primary CD4+ T cells

Agents that activate cannabinoid receptor pathways have been tested as treatments for cachexia, nausea or neuropathic pain in HIV-1/AIDS patients. The cannabinoid receptors (CB(1)R and CB(2)R) and the HIV-1 co-receptors, CCR5 and CXCR4, all signal via Gαi-coupled pathways. We hypothesized that drugs targeting cannabinoid receptors modulate chemokine co-receptor function and regulate HIV-1 infectivity. We found that agonism of CB(2)R, but not CB(1)R, reduced infection in primary CD4+ T cells following cell-free and cell-to-cell transmission of CXCR4-tropic virus. As this change in viral permissiveness was most pronounced in unstimulated T cells, we investigated the effect of CB(2)R agonism on to CXCR4-induced signaling following binding of chemokine or virus to the co-receptor. We found that CB(2)R agonism decreased CXCR4-activation mediated G-protein activity and MAPK phosphorylation. Furthermore, CB(2)R agonism altered the cytoskeletal architecture of resting CD4+ T cells by decreasing F-actin levels. Our findings suggest that CB(2)R activation in CD4+ T cells can inhibit actin reorganization and impair productive infection following cell-free or cell-associated viral acquisition of CXCR4-tropic HIV-1 in resting cells. Therefore, the clinical use of CB(2)R agonists in the treatment of AIDS symptoms may also exert beneficial adjunctive antiviral effects against CXCR4-tropic viruses in late stages of HIV-1 infection.     

CXCR7 mediated Giα independent activation of ERK and Akt promotes cell survival and chemotaxis in T cells

Chemokine receptors CXCR7 and CXCR4 bind to the same ligand stromal cell derived factor-1alpha (SDF-1α/CXCL12). We assessed the downstream signaling pathways mediated by CXCL12-CXCR7 interaction in Jurkat T cells. All experiments were carried out after functionally blocking the CXCR4 receptor. CXCL12, on binding CXCR7, induced phosphorylation of extra cellular regulated protein kinases (ERK 1/2) and Akt. Selective inhibition of each signal demonstrated that phosphorylated ERK 1/2 is essential for chemotaxis and survival of T cells whereas activation of Akt promotes only cell survival. Another interesting finding of this study is that CXCL12-CXCR7 interaction under normal physiological conditions does not activate the p38 pathway. Furthermore, we observed that the CXCL12 signaling via CXCR7 is Giα independent. Our findings suggest that CXCR7 promotes cell survival and does not induce cell death in T cells. The CXCL12 signaling via CXCR7 may be crucial in determining the fate of the activated T cells.     

Signaling domains of the interleukin 2 receptor

Interleukin (IL-)2 and its receptor (IL-2R) constitute one of the most extensively studied cytokine receptor systems. IL-2 is produced primarily by activated T cells and is involved in early T cell activation as well as in maintaining homeostatic immune responses that prevent autoimmunity. This review focuses on molecular signaling pathways triggered by the IL-2/IL-2R complex, with an emphasis on how the IL-2R physically translates its interaction with IL-2 into a coherent biological outcome. The IL-2R is composed of three subunits, IL-2Ralpha, IL-2Rbeta and gammac. Although IL-2Ralpha is an important affinity modulator that is essential for proper responses in vivo, it does not contribute to signaling due a short cytoplasmic tail. In contrast, IL-2Rbeta and gammac together are necessary and sufficient for effective signal transduction, and they serve physically to connect the receptor complex to cytoplasmic signaling intermediates. Despite an absolute requirement for gammac in signaling, the majority of known pathways physically link to the receptor via IL-2Rbeta, generally through phosphorylated cytoplasmic tyrosine residues. This review highlights work performed both in cultured cells and in vivo that defines the functional contributions of specific receptor subdomains-and, by inference, the specific signaling pathways that they activate-to IL-2-dependent biological activities.     

CD80 cytoplasmic domain controls localization of CD28, CTLA-4, and protein kinase Ctheta in the immunological synapse

The binding of costimulatory ligand CD80 to CD28 or CTLA-4 on T cells plays an important role in the regulation of the T cell response. We have examined the role of the cytoplasmic domain of CD80 in murine T cell costimulation and its organization in the immunological synapse (IS). Removal of CD80 cytoplasmic tail decreased its effectiveness in costimulating T cell proliferative response and early IL-2 production in response to agonist MHC-peptide complexes. Immunofluorescent study showed a decreased tailless CD80 accumulation in the IS of naive T cells. The two forms of CD80 accumulated differently at the IS; the tailless CD80 was colocalized with the TCR whereas the full-length CD80 was segregated from the TCR. In addition, we showed that CD80, CD28, and protein kinase Ctheta colocalized in the presence or absence of the CD80 cytoplasmic tail. Thus, the cytoplasmic tail of CD80 regulates its spatial localization at the IS and that of its receptors and T cell signaling molecules such as protein kinase Ctheta, and thereby facilitates full T cell activation.     

CXCL12 activation of CXCR4 regulates mucosal host defense through stimulation of epithelial cell migration and promotion of intestinal barrier integrity

Intestinal epithelial cell migration plays a key role in gastrointestinal mucosal barrier formation, enterocyte development, differentiation, turnover, wound healing, and adenocarcinoma metastasis. Chemokines, through engagement of their corresponding receptors, are potent mediators of directed cell migration and are critical in the establishment and regulation of innate and adaptive immune responses. The aim of this study was to define the role for the chemokine CXCL12 and its sole cognate receptor CXCR4 in regulating intestinal epithelial cell migration and to determine its impact on barrier integrity. CXCL12 stimulated the dose-dependent chemotactic migration of human T84 colonic epithelial cells. Epithelial cell migration was inhibited by CXCR4 neutralizing antibody, pertussis toxin, LY-294002, and PD-98059, thereby implicating Galpha(i), phosphatidylinositol 3-kinase (PI3-kinase), and the ERK1/2 MAP kinase pathways in CXCR4-specific signaling. CXCL12 was also shown to increase barrier integrity, as defined by transepithelial resistance and paracellular flux across differentiating T84 monolayers. To determine whether CXCL12 regulated epithelial restitution, we used the normal nontransformed intestinal epithelial cell-6 (IEC-6) wound healing model. By using RT-PCR, immunoblot analysis, and immunofluorescence microscopy, we first showed expression of both CXCR4 and its ligand by IEC-6 cells. We then demonstrated that CXCL12 activated comparable signaling mechanisms to stimulate epithelial migration in the absence of proliferation in wounded IEC-6 monolayers. Taken together, these data indicate that CXCL12 signaling via CXCR4 directs intestinal epithelial cell migration, barrier maturation, and restitution, consistent with an important mechanistic role for these molecules in mucosal barrier integrity and innate host defense.     

Quantitative phosphoproteomics of CXCL12 (SDF-1) signaling

CXCL12 (SDF-1) is a chemokine that binds to and signals through the seven transmembrane receptor CXCR4. The CXCL12/CXCR4 signaling axis has been implicated in both cancer metastases and human immunodeficiency virus type 1 (HIV-1) infection and a more complete understanding of CXCL12/CXCR4 signaling pathways may support efforts to develop therapeutics for these diseases. Mass spectrometry-based phosphoproteomics has emerged as an important tool in studying signaling networks in an unbiased fashion. We employed stable isotope labeling with amino acids in cell culture (SILAC) quantitative phosphoproteomics to examine the CXCL12/CXCR4 signaling axis in the human lymphoblastic CEM cell line. We quantified 4,074 unique SILAC pairs from 1,673 proteins and 89 phosphopeptides were deemed CXCL12-responsive in biological replicates. Several well established CXCL12-responsive phosphosites such as AKT (pS473) and ERK2 (pY204) were confirmed in our study. We also validated two novel CXCL12-responsive phosphosites, stathmin (pS16) and AKT1S1 (pT246) by Western blot. Pathway analysis and comparisons with other phosphoproteomic datasets revealed that genes from CXCL12-responsive phosphosites are enriched for cellular pathways such as T cell activation, epidermal growth factor and mammalian target of rapamycin (mTOR) signaling, pathways which have previously been linked to CXCL12/CXCR4 signaling. Several of the novel CXCL12-responsive phosphoproteins from our study have also been implicated with cellular migration and HIV-1 infection, thus providing an attractive list of potential targets for the development of cancer metastasis and HIV-1 therapeutics and for furthering our understanding of chemokine signaling regulation by reversible phosphorylation.     

CXCR7/CXCR4 heterodimer constitutively recruits beta-arrestin to enhance cell migration

G protein-coupled receptor hetero-oligomerization is emerging as an important regulator of ligand-dependent transmembrane signaling, but precisely how receptor heteromers affect receptor pharmacology remains largely unknown. In this study, we have attempted to identify the functional significance of the heteromeric complex between CXCR4 and CXCR7 chemokine receptors. We demonstrate that co-expression of CXCR7 with CXCR4 results in constitutive recruitment of β-arrestin to the CXCR4·CXCR7 complex and simultaneous impairment of G(i)-mediated signaling. CXCR7/CXCR4 co-expression also results in potentiation of CXCL12 (SDF-1)-mediated downstream β-arrestin-dependent cell signaling pathways, including ERK1/2, p38 MAPK, and SAPK as judged from the results of experiments using siRNA knockdown to deplete β-arrestin. Interestingly, CXCR7/CXCR4 co-expression enhances cell migration in response to CXCL12 stimulation. Again, inhibition of β-arrestin using either siRNA knockdown or a dominant negative mutant abrogates the enhanced CXCL12-dependent migration of CXCR4/CXCR7-expressing cells. These results show how CXCR7, which cannot signal directly through G protein-linked pathways, can nevertheless affect cellular signaling networks by forming a heteromeric complex with CXCR4. The CXCR4·CXCR7 heterodimer complex recruits β-arrestin, resulting in preferential activation of β-arrestin-linked signaling pathways over canonical G protein pathways. CXCL12-dependent signaling of CXCR4 and its role in cellular physiology, including cancer metastasis, should be evaluated in the context of potential functional hetero-oligomerization with CXCR7.     

G protein-coupled chemokine receptors induce both survival and apoptotic signaling pathways

Chemokine receptors are essential for triggering chemotaxis to immune cells; however, a number of them can also mediate death when engaged by nonchemokine ligands. When the chemokine receptor CXCR4 is engaged by stromal cell-derived factor (SDF1)alpha, it triggers cells to chemotax, and in some cell types such as neurons, causes cell death. To elucidate this dual and opposing receptor function, we have investigated whether CXCR4 activation by its chemokine SDF1alpha could lead to the simultaneous activation of both anti- and proapoptotic signaling pathways; the balance ultimately influencing cell survival. CXCR4 activation in CD4 T cells by SDF1alpha led to the activation of the prosurvival second messengers, Akt and extracellular signal-regulated protein kinase. Selective inhibition of each signal demonstrated that extracellular signal-regulated protein kinase is essential for mediating SDF1alpha-triggered chemotaxis but does not confer an antiapoptotic state. In contrast, Akt activation through CXCR4 by SDF1alpha interactions is necessary to confer resistance to apoptosis. The proapoptotic signaling pathway triggered by SDF1alpha-CXCR4 interaction involves the G(ialpha) protein-independent activation of the proapoptotic MAPK (p38). Furthermore, other chemokines and chemokine receptors also signal chemotaxis and proapoptotic effects via similar pathways. Thus, G(ialpha) protein-coupled chemokine receptors can function as death prone receptors and the balance between the above signaling pathways will ultimately mandate the fate of the activated cell.     

CXCR3 and IFN protein-10 in Pneumocystis pneumonia

We have previously shown that Tc1 CD8(+) T cells have in vitro and in vivo effector activity against Pneumocystis (PC) infection in mice. Because these cells have preferential expression of CXCR3, we investigated whether CXCR3 was required for host defense activity against PC. Mice deficient in CXCR3 but CD4(+) T cell intact, showed an initial delay but were able to clear the infectious challenge, indicating that CXCR3 signaling is not essential for clearance of PC. CD4-depleted mice had lower levels of monokine induced by IFN-gamma, IFN protein-10 (IP-10), and IFN-inducible T cell alpha-chemoattractant at day 7 of infection and are permissive to PC infection. Overexpression of IP-10 in the lungs by adenoviral gene transfer did not accelerate clearance of infection in control mice but accelerated clearance by day 28 in mice depleted of CD4(+) T cells. This effect was associated with increased recruitment of CD8(+) T to the lungs with higher CXCR3(+) expression levels and enhanced IFN-gamma secretion upon in vitro activation compared with control mice. These results indicate that the CXCR3 chemokines are part of the host defense response to PC, and that IP-10 can direct Tc1 CD8(+) T cell recruitment to the lungs and contribute to host defense against PC even in the absence of CD4(+) T cells.