The WHIM syndrome is a rare immunodeficiency disorder characterized by warts, hypogammaglobulinemia, infections, and myelokathexis. Dominant heterozygous mutations of the gene encoding CXCR4, a G-protein-coupled receptor with a unique ligand, CXCL12, have been associated with this pathology. We studied patients belonging to 3 different pedigrees. Two siblings inherited a CXCR4 mutation encoding a novel C-terminally truncated receptor. Two unrelated patients were found to bear a wild-type CXCR4 open reading frame. Circulating lymphocytes and neutrophils from all patients displayed similar functional alterations of CXCR4-mediated responses featured by a marked enhancement of G-protein-dependent responses. This phenomenon relies on the refractoriness of CXCR4 to be both desensitized and internalized in response to CXCL12. Therefore, the aberrant dysfunction of the CXCR4-mediated signaling constitutes a common biologic trait of WHIM syndromes with different causative genetic anomalies. Responses to other chemokines, namely CCL4, CCL5, and CCL21, were preserved, suggesting that, in clinical forms associated with a wild-type CXCR4 open reading frame, the genetic anomaly might target an effector with some degree of selectivity for the CXCL12/ CXCR4 axis. We propose that the sus- IntroductionThe CXC chemokine stromal cell-derived factor 1 (SDF-1/ CXCL12) 1,2 is the sole natural ligand for CXCR4, 3,4 a broadly expressed G-protein-coupled receptor (GPCR). 5 The unique, nonpromiscuous interaction between CXCL12 and CXCR4 is critically involved in the organogenesis of a number of phylogenetically distant animal species. [6][7][8][9][10][11] In addition, B-cell lymphopoiesis and bone marrow (BM) myelopoiesis are regulated by the CXCL12/ CXCR4 axis during embryogenesis. [12][13][14] In postnatal life, the CXCL12/CXCR4 couple controls the BM homing of CD34 ϩ cells and lymphocyte trafficking. [15][16][17][18] Besides the regulation of homeostatic processes, CXCR4 has been implicated in the development of infectious 3,19 and inflammatory diseases as well as tumor metastasis. [20][21][22][23] Recently, inherited heterozygous autosomal dominant mutations of the CXCR4 gene, which result in the truncation of the carboxyl-terminus (C-tail) of the receptor, were found to be associated with the WHIM syndrome. 24 This rare immunodeficiency disease is characterized by disseminated human papillomavirus (HPV)-induced warts, hypogammaglobulinemia, recurrent bacterial infections, and myelokathexis, a form of neutropenia associated with abnormal retention of mature neutrophils in the BM. [25][26][27] Patients with WHIM also exhibit a marked T-cell lymphopenia. The disorder is clinically and genetically heterogeneous, 28 since hypogammaglobulinemia and verrucosis were absent in some cases, 29 and individuals with isolated myelokathexis were found to be wild type for the CXCR4 gene. 24 However, the altered mechanism accounting for the pathogenesis of the WHIM syndrome not associated to CXCR4 mutations remains unknown. Here, we provide original...

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...

Activation of CXCR4 by the CXC chemokine stromal cell-derived factor-1 (SDF-1) requires interaction of the amino-terminal domains of both molecules. We report that proteinases released from either mononucleated blood cells or polymorphonuclear neutrophils degranulated by inflammatory stimuli generate an SDF-1 fragment that is deleted from amino-terminal residues Lys 1 -Pro 2 -Val 3 , as characterized by mass spectrometry analysis. The proteolyzed chemokine fails to induce agonistic functions and is unable to prevent the fusogenic capacity of CXCR4-tropic human immunodeficiency viruses. Furthermore, we observed that exposure of CXCR4-expressing cells to leukocyte proteinases results in the proteolysis of the extracellular aminoterminal domain of the receptor, as assessed by flow cytometry analysis and electrophoretic separation of immunoprecipitated CXCR4. Blockade of SDF-1 and CXCR4 proteolysis by the specific leukocyte elastase inhibitor, N-methoxysuccinyl-alanine-alanine-prolinevaline-chloromethyl ketone, identified elastase as the major enzyme among leukocyte-secreted proteinases that accounts for inactivation of both SDF-1 and CXCR4. Indeed, purified leukocyte elastase generated in either SDF-1 or CXCR4 a pattern of cleavage indistinguishable from that observed with leukocyte-secreted proteinases. Our findings suggest that elastase-mediated proteolysis of SDF-1/CXCR4 is part of a mechanism regulating their biological functions in both homeostatic and pathologic processes.

We have shown that the chemokine and HIV receptor CCR5 is palmitoylated on a cluster of cysteine residues located at the boundary between the seventh transmembrane region and the cytoplasmic tail. Single or combined substitutions of the three cysteines (Cys-321, Cys-323, and Cys-324) or incubation of wild-type CCR5-transfected cells with the palmitic acid analog 2-bromopalmitate prevented palmitoylation of the receptor. Moreover, failure of CCR5 to be palmitoylated resulted in both accumulation in intracellular stores and a profound decrease of membrane expression of the receptor. Upon metabolic labeling, kinetic experiments showed that the half-life of palmitoylation-deficient CCR5 is profoundly decreased. Bafilomycin A1, but not a specific proteasome inhibitor, prevented early degradation of palmitoylation-deficient CCR5 and promoted its accumulation in lysosomal compartments. Although membrane expression of the CCR5 mutant was diminished, the molecules reaching the membrane were still able to interact efficiently with the chemokine ligand MIP1␤ and remained able to function as HIV co-receptors. Thus we conclude that palmitoylation controls CCR5 expression through regulation of the life span of this receptor.G-protein-coupled receptors (GPCRs) 1 are formed by seven transmembrane hydrophobic ␣-helixes connected by extra-and intracellular loops. A glycosylated extracellular amino-terminal domain and a cytoplasmic carboxyl-terminal domain complete the structure of this superfamily of proteins, which transduces signals from a large and disparate number of extracellular ligands (1-3). Among GPCRs, chemokine receptors mediate the biological effects of chemokines that are structurally related to pro-inflammatory cytokines and are primarily involved in the control of constitutive and pathological migration of leukocytes (4, 5). However, a rapidly growing body of evidence shows that functions of chemokine receptors are not limited to locomotion and extends to a vast array of immune responses, organ and tissue development, as well as the pathogenesis of inflammatory, cardiovascular, and infectious diseases (6, 7).Two of these chemokine receptors, CXCR4 and CCR5, have been identified as co-receptors that allow efficient entry of HIV into CD4ϩ host cells (8). Upon binding to CD4, the interaction of HIV isolates with CXCR4 or CCR5 (X4 and R5 isolates, respectively) relies on the structure of the HIV envelope (Env) glycoprotein, gp120 subunit. The binding of the HIV Env complex (gp120/gp41 subunits) to the receptors CD4 and CXCR4 or CCR5 initiate conformational changes in the gp120/gp41 oligomer, leading to membrane fusion and, ultimately, internalization of the viral capsid into the host cell (9, 10).CCR5 plays a critical role in the transmission and early propagation of HIV-1 (11-14). Thus, R5 isolates predominate for years after primo-infection and are very often the only variants detected in AIDS patients. Identification of a CCR5 gene polymorphism results in expression of severely truncated molecules that fail to rea...

The contribution of raft domains to human immunodeficiency virus (HIV) 1 entry was assessed. In particular, we asked whether the CD4 and CCR5 HIV-1 receptors need to associate with sphingolipid-enriched, detergent-resistant membrane domains (rafts) to allow viral entry into primary and T-cell lines. Based on Triton X-100 solubilization and confocal microscopy, CD4 was shown to distribute partially to rafts. In contrast, CCR5 did not associate with rafts and localized in nonraft plasma membrane domains. HIV-1-receptor partitioning remained unchanged upon viral adsorption, suggesting that viral entry probably takes place outside rafts. To directly investigate this possibility, we targeted CD4 to nonraft domains of the membrane by preventing CD4 palmitoylation and interaction with p56 lck . Directed mutagenesis of both targeting signals significantly prevented association of CD4 with rafts, but did not suppress the HIV-1 receptor function of CD4. Collectively, these results strongly suggest that the presence of HIV-1 receptors in rafts is not required for viral infection. We show, however, that depleting plasma membrane cholesterol inhibits HIV-1 entry. We therefore propose that cholesterol modulates the HIV-1 entry process independently of its ability to promote raft formation.

CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor that governs migration of leukocytes and serves as a coreceptor for the R5 tropic strains of human immunodeficiency virus (HIV). CCR5-mediated signaling in response to CC chemokines relies on G protein activation. Desensitization, which rapidly turns off G protein-dependent signaling, involves phosphorylation of CCR5 that promotes interaction of the receptor with ␤-arrestins for endocytosis. Whether coupling to G proteins, desensitization, and endocytosis of CCR5 require the same structural determinants remains a matter of investigation. Here, we show that CCR5 displayed agonist-independent coupling to G proteins. This constitutive activity of the receptor was abrogated by TAK779, a nonpeptidic CCR5 ligand that inhibits HIV infection and was found to depend on the integrity of the AspArg-Tyr (DRY) motif. Changing Arg-126 by the neutral residue Asn (R126N-CCR5 mutant) abolished CCR5-mediated activation of G proteins, either constitutively or in response to agonists. In contrast, R126N-CCR5 not only retained agonist-promoted phosphorylation and ␤-arrestin-dependent endocytosis but also displayed a higher basal phosphorylation than wild-type CCR5. Expression of ␤-arrestin in R126N-CCR5-expressing cells resulted in receptor down-regulation, thereby suggesting that R126N-CCR5 spontaneously interacts with ␤-arrestins. However, although expression of ␤-arrestin favored wild-type CCR5-mediated chemotaxis, it failed to promote migration of cells expressing R126N-CCR5. Overall, these data indicate that structural requirements for CCR5-mediated activation of G proteins, albeit not involved in receptor desensitization and internalization, are needed for ␤-arrestin-mediated chemotaxis. These results have implications for how distinct biological responses of CCR5 might rely on a different set of receptor conformations.CCR5 is a G protein-coupled heptahelical receptor (GPCR) that initiates intracellular signaling in response to CC chemokines, including CCL3/MIP-1␣, CCL4/MIP-1␤, CCL5/RANTES, and CCL8/monocyte chemoattractant protein 2. CCR5 is involved in the recruitment of leukocytes to the site of inflammation and is also associated to a number of pathological disorders, from inflammatory diseases to AIDS (Proudfoot, 2002). In the present work, we deal with the molecular mechanisms that underlie CCR5 conformational changes and functions.

The molecular determinants underlying the failure of axons to regenerate in the CNS after injury were studied in an in vitro model of astrogliosis and neuronal coculture. Mechanically lesioned neuron-astrocyte mouse cortical cocultures were treated with antisense glial fibrillary acidic protein (GFAP)-mRNA in order to inhibit the formation of gliofilaments that occurs in response to injury. This inhibition relieves the blockage of neuron migration and neuritic outgrowth observed after lesion, and migrating neurons reappeared, supported by a laminin-labeled extracellular network (permissive conditions). We then questioned the relationship between this permissivity and laminin production. Follow-up studies on the concentration of laminin indicated that, after antisense treatment, the laminin level was increased in the cocultures and was under the control of astrocyte-neuron interactions. The addition of exogenous laminin favored neuronal migration and neurite outgrowth, whereas neutralizing laminin bioavailability with antibodies recognizing the astroglial laminin resulted in an inhibition of both neuronal access to the lesion site and neurite outgrowth, suggesting an active role for laminin in the permissive process. This permissive process could be associated with modulation of extracellular matrix (ECM) molecule degradation by proteinases. Among the latter, matrix metalloproteinases (MMPs) are involved in the breakdown of the ECM component. Our investigation showed a net decrease of the matrix metalloproteinase MMP-2 expression and activity and an increase of its endogenous inhibitor TIMP-2 expression. Both proteins associated with permissivity should be involved in the laminin stabilization and cell-matrix interactions. High levels of laminin and laminin bioavailability, consequent to a reduction in astrogliosis, may be important permissive elements for neuronal migration and neurite outgrowth postlesion.

Circulating matrix metalloproteinases (MMP-9) correlates with disease severity in Guillain-Barré syndrome (GBS). MMP-9 likely represents an important molecule in the pathogenesis of GBS and therefore could represent an interesting therapeutic target.