3,4-Diamino-2,5-thiadiazole-1-oxides as potent CXCR2/CXCR1 antagonists

A series of novel and potent 3,4-diamino-2,5-thiadiazole-1-oxides were prepared and found to show excellent binding affinities for CXCR2 and CXCR1 receptors and excellent inhibitory activity of Gro-alpha and IL-8 mediated in vitro hPMN MPO release of CXCR2 and CXCR1 expressing cell lines. On the other hand, a closely related 3,4-diamino-2,5-thiadiazole-dioxide did not show functional activity despite its excellent binding affinities for CXCR2 and CXCR1 in membrane binding assays. A detailed SAR has been discussed in these two closely related structures.     

Cysteine residues are critical for chemokine receptor CXCR2 functional properties

We examined the role of cysteine (Cys) residues present in chemokine receptor CXCR2 for proper surface expression, dimerization, signaling, and chemotaxis. To address this issue, serine or leucine residues were substituted for Cys, generating nine CXCR2 mutants transiently expressed in HEK cells. Single substitution of Cys residues present in the three extracellular loops (C119L, C196L, C286S) or in the seventh-transmembrane (TM) domain (C308L) abolished CXCL8 agonist binding, while no Cys substitution abolished surface receptor expression. We have previously demonstrated that CXCR2 dimerizes under reducing conditions, due to hydrophobic interactions that involve TM3 regions, and here we show that the dimer/monomer CXCR2 ratio drastically increases when analyzed under non-reducing conditions. We report that none of the Cys-deficient CXCR2 mutants abolishes receptor dimerization, demonstrating that Cys-Cys bonds are not the exclusive determinant of CXCR2 dimerization. Furthermore, both wt- and Cys-mutated CXCR2 dimers are expressed at the cell surface, indicating that receptor dimers are efficiently transferred at the plasma membrane. We also show that every Cys substitution in CXCR2, including those that still bind CXCL8, results in an impairment of receptor activity, analyzed as cell chemotaxis and intracellular signaling, suggesting that some structural requirement is likely fulfilled by Cys presence.     

Intracellular cross-talk between the GPCR CXCR1 and CXCR2: role of carboxyl terminus phosphorylation sites

In the present study, we used the human chemokine receptors CXCR1 and CXCR2 as a model system for the study of intracellular cross-talk between two closely related G protein-coupled receptors (GPCR). In cells expressing either CXCR1 or CXCR2, exposure to the CXCL8 ligand resulted in prominent reduction in cell surface expression of the receptors. We have shown previously that the reduction in cell surface expression of CXCR1 and CXCR2, to be termed herein "down-regulation", is significantly lower in cells expressing both receptors together. Now we show that reduced receptor down-regulation was specific to the CXCR1+CXCR2 pair. Also, CXCR2 carboxyl terminus phosphorylation sites were required for inducing inhibition of CXCR1 down-regulation, and vice versa. Accordingly, phosphorylation of CXCR2 carboxyl terminus domain was intact when expressed together with CXCR1. Moreover, specific carboxyl terminus phosphorylation sites on each of the wild type receptors protected them from more severe inhibition of down-regulation, induced by joint expression with the other receptor. When concomitantly expressed, CXCR1 and CXCR2 were impaired in recycling to the plasma membrane, despite their undergoing intact dephosphorylation. Overall, we show that cross-talk between two GPCR is manifested by impairment of their intracellular trafficking, primarily of ligand-induced down-regulation, via carboxyl terminus phosphorylation sites.     

Arrestin-2 interacts with the ubiquitin-protein isopeptide ligase atrophin-interacting protein 4 and mediates endosomal sorting of the chemokine receptor CXCR4

The chemokine receptor CXCR4 is rapidly targeted for lysosomal degradation by the E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4). Although it is known that AIP4 mediates ubiquitination and degradation of CXCR4 and that perturbations in these events contribute to disease, the mechanisms mediating AIP4-dependent regulation of CXCR4 degradation remain poorly understood. Here we show that AIP4 directly interacts with the amino-terminal half of nonvisual arrestin-2 via its WW domains. We show that depletion of arrestin-2 by small interfering RNA blocks agonist-promoted degradation of CXCR4 by preventing CXCR4 trafficking from early endosomes to lysosomes. Surprisingly, CXCR4 internalization and ubiquitination remain intact, suggesting that the interaction between arrestin-2 and AIP4 is not required for ubiquitination of the receptor at the plasma membrane but perhaps for a later post-internalization event. Accordingly, we show that activation of CXCR4 promotes the interaction between AIP4 and arrestin-2 that is consistent with a time when AIP4 co-localizes with arrestin-2 on endocytic vesicles. Taken together, our data suggest that the AIP4.arrestin-2 complex functions on endosomes to regulate sorting of CXCR4 into the degradative pathway.     

On the mechanism and significance of ligand-induced internalization of human neutrophil chemokine receptors CXCR1 and CXCR2

It is well established that leukocyte chemotactic receptors, a subset of G protein-coupled receptors, undergo endocytosis after stimulation by ligand. However, the significance of this phenomenon to cell motility and other important leukocyte functions induced by chemoattractants has not been clearly defined. Here we show that in primary human neutrophils, the threshold levels of agonist required for endocytosis of the chemotactic receptors CXCR1 and CXCR2 were approximately 10-fold or higher than those needed for maximal chemotactic and calcium flux responses. Moreover, when stimulated by agonists at concentrations that are high enough for chemotaxis but too low for receptor endocytosis, neutrophil CXCR1 and CXCR2 could be reactivated in response to repeated application of the same agonist. Both receptors were excluded from Triton X-100-insoluble lipid rafts, and at high agonist concentrations were rapidly endocytosed by a clathrin/rab5/dynamin-dependent pathway. These data support the conclusion that neutrophil migration in response to CXCR1 or CXCR2 agonists is not dependent on endocytosis of CXCR1 or CXCR2. Rather than being integral to the process of cell migration, receptor endocytosis may be a terminal stop signal when cells reach the focus of inflammation where the chemoattractant concentrations are the highest.     

The role of CXCR2 activity in the contact hypersensitivity response in mice

The recruitment of polymorphonuclear neutrophil leukocytes (PMN) into a challenge site, and their subsequent activation, are thought to play a role in the elicitation of the contact hypersensitivity (CHS) response. The present study investigated the role played by CXCR2 activity in tissue PMN infiltration and subsequent triggering of CHS. Our results show that the cutaneous infiltration by PMN, induced by hapten challenge was dramatically inhibited in sensitized, CXCR2-deficient (CXCR2(-/-)) mice. Inhibition of PMN recruitment into the hapten-challenged ears of CXCR2(-/-) mice was associated with a consistent reduction of the CHS response (ear swelling) in CXCR2(-/-) mice as compared with that observed in neutropenic, wild-type (CXCR2(+/+)) mice. Prevention of skin PMN infiltration and the ear swelling response by the absence of functional CXCR2 was observed regardless of the hapten used. These data clearly suggest that CXCR2 activity plays an essential role in mediating cutaneous recruitment and activation of PMN, and thus indirectly regulates recruitment of hapten-primed T cells into challenge sites, with the subsequent elicitation of the CHS response. The role played by CXCR2 activity in the CHS response provides the rationale for testing CXCR2 inhibitors as a new therapeutic approach to skin diseases.     

A potential role of the GRO-α/CXCR2 system in Sjögren’s syndrome: regulatory effects of pro-inflammatory cytokines

Chemokines, small pro-inflammatory cytokines, are involved in migration of inflammatory cells in inflamed tissues and recent studies established their role in angiogenesis, hematopoiesis, cancer and autoimmune conditions. Growth related oncogene-alpha (GRO-α), a member of the CXC chemokine family, and its receptor CXCR2 are involved in the inflammatory processes. Since there is no previous report that supports a possible role of GRO-α/CXCR2 receptor complex during inflammation and neovascularization existing in the autoimmune disease Sjögren’s syndrome (SS), in this study, we examined CXCR2 and its ligand GRO-α expression in SS tissues. Immunohistochemistry revealed that GRO-α and its receptor CXCR2 were expressed at high levels in diseased tissues compared to healthy controls. In addition, human salivary gland epithelial cells (SGEC) cultures were submitted to a pro-inflammatory microenvironment using cytokines IL-6 and TNF-α in order to demonstrate that CXCR2 may change its initial expression pattern to another under inflammatory condition. The data show an increased expression of CXCR2 depending on the inflammatory cytokine used in culture in a time-dependent manner. Furthermore, silencing of the pro-angiogenic chemokine GRO-α is proportionally correlated with decreased expression of CXCR2 in pro-inflammatory cytokine-stimulated SGEC indicating the GRO-α/CXCR2 complex as a novel therapeutic target for the chronic inflammatory disease Sjögren’s syndrome.     

Expression of CXCL4 in microglia in vitro and in vivo and its possible signaling through CXCR3

Signaling through chemokine receptor CXCR3 in the brain has been implicated in various brain diseases, as CXCR3 and its ligands are found under these conditions. Recently, a new chemokine ligand for CXCR3 was reported. In humans, an alternatively spliced variant of CXCR3 expressed on microvascular endothelial cells, named CXCR3b, was shown to bind CXCL4. In the periphery, the cellular expression and functions of CXCL4 are well described but in the brain its expression and function are unknown. Here, we show that brain microglia are a cellular source of CXCL4 in vitro and in vivo under neurodegenerating conditions. Microglial migration induced by CXCL4 is absent in CXCR3-deficient microglia, indicating a role of CXCR3. CXCL4 furthermore attenuates lipopolysaccharide-induced microglial phagocytosis and nitric oxide production in microglia and BV-2 cells. Based on these findings, it is proposed that locally released CXCL4 may control microglia responses.     

Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease

Interleukin-8 (IL-8) activates neutrophils via the chemokine receptors CXCR1 and CXCR2. However, the airways of individuals with cystic fibrosis are frequently colonized by bacterial pathogens, despite the presence of large numbers of neutrophils and IL-8. Here we show that IL-8 promotes bacterial killing by neutrophils through CXCR1 but not CXCR2. Unopposed proteolytic activity in the airways of individuals with cystic fibrosis cleaved CXCR1 on neutrophils and disabled their bacterial-killing capacity. These effects were protease concentration-dependent and also occurred to a lesser extent in individuals with chronic obstructive pulmonary disease. Receptor cleavage induced the release of glycosylated CXCR1 fragments that were capable of stimulating IL-8 production in bronchial epithelial cells via Toll-like receptor 2. In vivo inhibition of proteases by inhalation of alpha1-antitrypsin restored CXCR1 expression and improved bacterial killing in individuals with cystic fibrosis. The cleavage of CXCR1, the functional consequences of its cleavage, and the identification of soluble CXCR1 fragments that behave as bioactive components represent a new pathophysiologic mechanism in cystic fibrosis and other chronic lung diseases.     

Novel roles for the E3 ubiquitin ligase atrophin-interacting protein 4 and signal transduction adaptor molecule 1 in G protein-coupled receptor signaling

The CXCL12/CXCR4 signaling axis plays an important role in human health and disease; however, the molecular mechanisms mediating CXCR4 signaling remain poorly understood. Ubiquitin modification of CXCR4 by the E3 ubiquitin ligase AIP4 is required for lysosomal sorting and degradation, which is mediated by the endosomal sorting complex required for transport (ESCRT) machinery. CXCR4 sorting is regulated by an interaction between endosomal localized arrestin-2 and STAM-1, an ESCRT-0 component. Here, we report a novel role for AIP4 and STAM-1 in regulation of CXCR4 signaling that is distinct from their function in CXCR4 trafficking. Depletion of AIP4 and STAM-1 by siRNA caused significant inhibition of CXCR4-induced ERK-1/2 activation, whereas overexpression of these proteins enhanced CXCR4 signaling. We further show that AIP4 and STAM-1 physically interact and that the proline-rich region in AIP4 and the SH3 domain in STAM-1 are essential for the interaction. Overexpression of an AIP4 catalytically inactive mutant and a mutant that shows poor binding to STAM-1 fails to enhance CXCR4-induced ERK-1/2 signaling, as compared with wild-type AIP4, suggesting that the interaction between AIP4 and STAM-1 and the ligase activity of AIP4 are essential for ERK-1/2 activation. Remarkably, a discrete subpopulation of AIP4 and STAM-1 resides in caveolar microdomains with CXCR4 and appears to mediate ERK-1/2 signaling. We propose that AIP4-mediated ubiquitination of STAM-1 in caveolae coordinates activation of ERK-1/2 signaling. Thus, our study reveals a novel function for ubiquitin in the regulation of CXCR4 signaling, which may be broadly applicable to other G protein-coupled receptors.     

The carboxyl-terminal PDZ ligand motif of chemokine receptor CXCR2 modulates post-endocytic sorting and cellular chemotaxis

Adaptor protein interaction with specific peptide motifs found within the intracellular, carboxyl terminus of chemokine receptor CXCR2 has been shown to modulate intracellular trafficking and receptor function. Efficient ligand-induced internalization of this receptor is dependent on the binding of adaptor protein 2 to the specific LLKIL motif found within the carboxyl terminus (1). In this study we show that the carboxyl-terminal type 1 PDZ ligand motif (-STTL) of CXCR2 plays an essential role in both proper intracellular receptor trafficking and efficient cellular chemotaxis. First, we show that CXCR2 is sorted to and degraded in the lysosome upon long-term ligand stimulation. We also show that receptor degradation is not dependent upon receptor ubiquitination, but is instead modulated by the carboxyl-terminal type I PDZ ligand of CXCR2. Deletion of this ligand results in increased degradation, earlier co-localization with the lysosome, and enhanced sorting to the Rab7-positive late endosome. We also show that deletion of this ligand effects neither receptor internalization nor receptor recycling. Furthermore, we demonstrate that deletion of the PDZ ligand motif results in impaired chemotactic response. The data presented here demonstrate that the type I PDZ ligand of CXCR2 acts to both delay lysosomal sorting and facilitate proper chemotactic response.     

Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration,activation, and regulation

IL-8 (or CXCL8) activates the receptors CXCR1 (IL-8RA) and CXCR2 (IL-8RB) to induce chemotaxis in leukocytes, but only CXCR1 mediates cytotoxic and cross-regulatory signals. This may be due to the rapid internalization of CXCR2. To investigate the roles of the intracellular domains in receptor regulation, wild-type, chimeric, phosphorylation-deficient, and cytoplasmic tail (C-tail) deletion mutants of both receptors were expressed in RBL-2H3 cells and studied for cellular activation, receptor phosphorylation, desensitization, and internalization. All but one chimeric receptor bound IL-8 and mediated signal transduction, chemotaxis, and exocytosis. Upon IL-8 activation, the chimeric receptors underwent receptor phosphorylation and desensitization. One was resistant to internalization, yet it mediated normal levels of beta-arrestin 2 (beta arr-2) translocation. The lack of internalization by this receptor may be due to its reduced association with beta arr-2 and the adaptor protein-2 beta. The C-tail-deleted and phosphorylation-deficient receptors were resistant to receptor phosphorylation, desensitization, arrestin translocation, and internalization. They also mediated greater phosphoinositide hydrolysis and exocytosis and sustained Ca(2+) mobilization, but diminished chemotaxis. These data indicate that phosphorylation of the C-tails of CXCR1 and CXCR2 are required for arrestin translocation and internalization, but are not sufficient to explain the rapid internalization of CXCR2 relative to CXCR1. The data also show that receptor internalization is not required for chemotaxis. The lack of receptor phosphorylation was correlated with greater signal transduction but diminished chemotaxis, indicating that second messenger production, not receptor internalization, negatively regulates chemotaxis.     

CXC chemokines and their receptors: a case for a significant biological role in cutaneous wound healing

Wound healing requires a complex series of reactions and interactions among cells and their mediators, resulting in an overlapping series of events including coagulation, inflammation, epithelialization, formation of granulation tissue, matrix and scar formation. Cytokines and chemokines promote inflammation, angiogenesis, facilitate the passage of leukocytes from circulation into the tissue, and contribute to the regulation of epithelialization. They integrate inflammatory events and reparative processes that are important for modulating wound healing. Thus both cytokines and chemokines are important targets for therapeutic intervention. The chemokine-mediated regulation of angiogenesis is highly sophisticated, fine tuned, and involves pro-angiogenic chemokines, including CXCL1-3, 5-8 and their receptors, CXCR1 and CXCR2. CXCL1 and CXCR2 are expressed in normal human epidermis and are further induced during the wound healing process of human burn wounds, especially during the inflammatory, epithelialization and angiogenic processes. Human skin explant studies also show CXCR2 is expressed in wounded keratinocytes and Th/1/Th2 cytokine modulation of CXCR2 expression correlates with proliferation of epidermal keratinocytes. Murine excision wound healing, chemical burn wounds and skin organ culture systems are valuable models for examining the role of inflammatory cytokines and chemokines in wound healing.     

Substitutions in a homologous region of extracellular loop 2 of CXCR4 and CCR5 alter coreceptor activities for HIV-1 membrane fusion and virus entry

CXCR4 and CCR5 are the principal coreceptors for human immunodeficiency virus type-1 (HIV-1) infection. Previously, mutagenesis of CXCR4 identified single amino acid changes that either impaired CXCR4's coreceptor activity for CXCR4-dependent (X4) isolate envelope glycoproteins (Env) or expanded its activity, allowing it to serve as a functional coreceptor for CCR5-dependent (R5) isolates. The most potent of these point mutations was an alanine substitution for the aspartic acid residue at position 187 in extracellular loop 2 (ecl-2), and here we show that this mutation also permits a variety of primary R5 isolate Envs, including those of other subtypes (clades), to employ it as a coreceptor. We also examined the corresponding region of CCR5 and demonstrate that the substitution of the serine residue in the homologous ecl-2 position with aspartic acid impairs CCR5 coreceptor activity for isolates across several clades. These results highlight a homologous and critical element in ecl-2, of both the CXCR4 and CCR5 molecules, for their respective coreceptor activities. Charge elimination expands CXCR4 coreceptor activity, while a similar charge introduction can destroy the coreceptor function of CCR5. These findings provide further evidence that there are conserved elements in both CXCR4 and CCR5 involved in coreceptor function.