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Philip M Murphy - One of the best experts on this subject based on the ideXlab platform.

  • the cxc chemokines growth regulated oncogene gro α groβ groγ neutrophil activating peptide 2 and epithelial cell derived neutrophil activating peptide 78 are potent agonists for the type b but not the type a human Interleukin 8 Receptor
    Journal of Biological Chemistry, 1996
    Co-Authors: Sunil K. Ahuja, Philip M Murphy
    Abstract:

    Abstract Interleukin-8 (IL-8), growth-related oncogene (GRO) α, GROβ, GROγ, neutrophil-activating peptide-2 (NAP-2), epithelial cell-derived neutrophil activating peptide- 78 (ENA-78), and granulocyte chemoattractant protein-2 are potent neutrophil chemoattractants 40-90% identical in amino acid sequence that comprise a subgroup of human CXC chemokines defined by the conserved sequence motif glutamic acid-leucine-arginine (ELR). Two human chemotactic Receptor subtypes for IL-8, named IL-8 Receptors (IL8R) A and B, have been cloned. They are 78% identical in amino acid sequence, coexpressed in neutrophils, and distinguished by their different selectivities for GROα and NAP-2. Their selectivity for other ELR+ CXC chemokines has not been previously reported. By measuring calcium flux in human embryonic kidney 293 cells transfected with plasmids encoding IL8RA or IL8RB, we have now defined Receptor selectivity for GROβ, GROγ, and ENA-78. The rank order of agonist potency, based on inspection of the mean effective concentration values (EC50), for IL8RB was GROγ (1 nM) > IL-8 (4 nM) ∼ GROα (5 nM) ∼ GROβ (4 nM) ∼ NAP-2 (7 nM) > ENA-78 (11 nM), and for IL8RA was IL-8 (4 nM) >>> ENA-78 (40 nM) ∼ NAP-2 (45 nM) > GROα (63 nM)∼GROγ (65 nM) ≫ GROβ. The maximal response of IL8RA to IL-8 was at least 2-fold greater than the other five chemokines. All six agonists for IL8RB competed for high affinity 125I-IL-8, -GROα, -NAP-2, and -ENA-78 binding sites at IL8RB. GROα, GROβ, GROγ, NAP-2, and ENA-78 competed weakly for the high affinity IL-8 binding site at IL8RA. Thus, IL8RA and IL8RB are both highly selective for IL-8 and have similar sequences but differ dramatically in their selectivity for all other ELR+ CXC chemokines tested. These findings have important implications for developing novel neutrophil-specific anti-inflammatory drugs directed against the CXC chemokine signaling system.

  • cxc chemokines bind to unique sets of selectivity determinants that can function independently and are broadly distributed on multiple domains of human Interleukin 8 Receptor b determinants of high affinity binding and Receptor activation are distinct
    Journal of Biological Chemistry, 1996
    Co-Authors: Sunil K. Ahuja, Jennifer C Lee, Philip M Murphy
    Abstract:

    Abstract Human Interleukin-8 Receptors A (IL-8RA) and B (IL-8RB) are seven-transmembrane domain (TMD) neutrophil chemokine Receptors with similar sequences (77% amino acid identity) and similar G protein selectivity, but markedly different selectivity for CXC chemokines. IL-8RB is selective for IL-8, growth-related oncogene α (GROα) and neutrophil-activating peptide-2 (NAP-2), whereas IL-8RA is selective only for IL-8. To identify selectivity determinants, we made eight chimeric Receptors exchanging: 1) the three main regions of sequence divergence between IL-8RA and IL-8RB (the N-terminal segment before TMD1, the region from TMD4 to the end of the second extracellular (e2) loop, and the C-terminal tail), and 2) the N-terminal segment of CC chemokine Receptor 1, which does not bind CXC chemokines. Chimeras were tested by direct I-IL-8, I-GROα, and I-NAP-2 binding, heterologous competition binding, and calcium flux assays using human embryonic kidney 293 cells stably transfected with Receptor DNAs. The following results were obtained: 1) chimeric Receptors had binding sites for IL-8, GROα and NAP-2 distinct from those on IL-8RA and IL-8RB; 2) IL-8, GROα and NAP-2 bound to overlapping but distinct sites that mapped differentially to multiple domains on IL-8RB; 3) high affinity radioligand binding and high agonist potency were separable functions for IL-8, GROα and NAP-2, suggesting that the determinants of high affinity binding may not be critical for Receptor activation; and 4) determinants of GROα and NAP-2 selectivity were found in both the N-terminal segment before TMD1 and the region from TMD4 to the end of the e2 loop of IL-8RB, and functioned independently of each other. Stated reciprocally, the N-terminal segment of IL-8RA was not a dominant selectivity determinant. These data suggest that both narrow and broad spectrum chemokine antagonists can be developed to block functions mediated by IL-8RB.

  • Molecular piracy of mammalian Interleukin-8 Receptor type B by herpesvirus saimiri.
    Journal of Biological Chemistry, 1993
    Co-Authors: Sunil K. Ahuja, Philip M Murphy
    Abstract:

    Abstract Viruses are known to acquire and modify the genes of their hosts to attain a survival advantage in the host environment. Herpesvirus saimiri (HVS) is a T-lymphotropic virus that causes fatal lymphoproliferative diseases in several non-human primates. The gene ECRF3 of HVS was most likely acquired from a primate host. ECRF3 encodes a putative seven-transmembrane-domain Receptor that is remotely related (approximately 30% amino acid identity) to the known mammalian alpha and beta chemokine Receptors, namely Interleukin-8 Receptor (IL8R) types A and B and the MIP-1 alpha/RANTES Receptor, respectively. Chemokines regulate the trafficking, activation, and, in some cases, proliferation of myeloid and lymphoid cell types. We now show that ECRF3 encodes a functional Receptor for the alpha chemokines IL-8, GRO/melanoma growth stimulatory activity (MGSA), and NAP-2 but not for beta chemokines, a specificity identical to that of IL8RB. Paradoxically, IL8RA shares 77% amino acid identity with IL8RB but is not a Receptor for GRO/MGSA or NAP-2. This is the first functional characterization of a viral seven-transmembrane-domain Receptor. It suggests a novel role for alpha chemokines in the pathogenesis of HVS infection by transmembrane signaling via the product of ECRF3.

  • molecular evolution of the human Interleukin 8 Receptor gene cluster
    Nature Genetics, 1992
    Co-Authors: Sunil K. Ahuja, Tayfun Ozcelik, Athena Milatovitch, Uta Francke, Philip M Murphy
    Abstract:

    lnterleukin–8 (IL–8) is the prototype for a family of at least eight neutrophil chemoattractants whose genes map to human chromosome 4q13–q21. Two human IL–8 Receptors, IL8RA and IL8RB, are known from cDNA cloning; IL8RA is a promiscuous Receptor for at least two other related ligands, GROα and NAP–2. We now report cloning of the genes for IL8RA, IL8RB and a recently inactivated pseudogene of Receptor A (IL8RAP). These form a cluster of only three genes in the superfamily of G protein–coupled Receptors (GPCRs) and map to 2q34–q35. The revolutionary diversity displayed by the IL–8 ligand–Receptor complex — ligand promiscuity for IL–8, Receptor promiscuity for IL8RA, gene duplication for both ligands and Receptors and gene extinction in the case of IL8RAP — is unprecedented for the GPCR superfamily.

  • cloning of complementary dna encoding a functional human Interleukin 8 Receptor
    Science, 1991
    Co-Authors: Philip M Murphy, H L Tiffany
    Abstract:

    Interleukin-8 (IL-8) is an inflammatory cytokine that activates neutrophil chemotaxis, degranulation, and the respiratory burst. Neutrophils express Receptors for IL-8 that are coupled to guanine nucleotide-binding proteins (G proteins); binding of IL-8 to its Receptor induces the mobilization of intracellular calcium stores. A cDNA clone from HL-60 neutrophils, designated p2, has now been isolated that encodes a human IL-8 Receptor. When p2 is expressed in oocytes from Xenopus laevis, the oocytes bind 125I-labeled IL-8 specifically and respond to IL-8 by mobilizing calcium stores with an EC50 of 20 nM. This IL-8 Receptor has 77% amino acid identity with a second human neutrophil Receptor isotype that binds IL-8 with higher affinity. It also exhibits 69% amino acid identity with a protein reported to be an N-formyl peptide Receptor from rabbit neutrophils, but less than 30% identity with all other known G protein-coupled Receptors, including the human N-formyl peptide Receptor.

Javier Navarro - One of the best experts on this subject based on the ideXlab platform.

  • Novel Down-regulatory Mechanism of the Surface Expression of the Vasopressin V2 Receptor by an Alternative Splice Receptor Variant
    Journal of Biological Chemistry, 2004
    Co-Authors: Jose Sarmiento, Carolina C. Añazco, Danae M. Campos, Gregory N. Prado, Javier Navarro, Carlos B. Gonzalez
    Abstract:

    Abstract In rat kidney, two alternatively spliced transcripts are generated from the V2 vasopressin Receptor gene. The large transcript (1.2 kb) encodes the canonical V2 Receptor, whereas the small transcript encodes a splice variant displaying a distinct sequence corresponding to the putative seventh transmembrane domain and the intracellular C terminus of the V2 Receptor. This work showed that the small spliced transcript is translated in the rat kidney collecting tubules. However, the protein encoded by the small transcript (here called the V2b splice variant) is retained inside the cell, in contrast to the preferential surface distribution of the V2 Receptor (here called the V2a Receptor). Cells expressing the V2b splice variant do not exhibit binding to 3H-labeled vasopressin. Interestingly, we found that expression of the splice variant V2b down-regulates the surface expression of the V2a Receptor, most likely via the formation of V2a·V2b heterodimers as demonstrated by co-immunoprecipitation and fluorescence resonance energy transfer experiments between the V2a Receptor and the V2b splice variant. The V2b splice variant would then be acting as a dominant negative. The effect of the V2b splice variant is specific, as it does not affect the surface expression of the G protein-coupled Interleukin-8 Receptor (CXCR1). Furthermore, the sequence encompassing residues 242–339, corresponding to the C-terminal domain of the V2b splice variant, also down-regulates the surface expression of the V2a Receptor. We suggest that some forms of nephrogenic diabetes insipidus are due to overexpression of the splice variant V2b, which could retain the wild-type V2a Receptor inside the cell via the formation of V2a·V2b heterodimers.

  • role of the c terminus of the Interleukin 8 Receptor in signal transduction and internalization
    Journal of Biological Chemistry, 1996
    Co-Authors: Gregory N. Prado, Hiroyuki Suzuki, Nancy Wilkinson, Beth Cousins, Javier Navarro
    Abstract:

    Interleukin 8 (IL-8) is a potent neutrophil chemoattractant and activator. Two IL-8 Receptor subtypes, A and B, are expressed in neutrophils. In this work, we analyzed the role of the C terminus domain of the IL-8 Receptor on the signal transduction and Receptor internalization mechanisms. The IL-8 Receptor A was tagged with an epitope corresponding to the monoclonal antibody 1D4 to monitor the localization of the IL-8 Receptor. We demonstrated IL-8-dependent Receptor internalization by monitoring the density of surface 125I-labeled IL-8 binding sites and by immunofluorescence microscopy. Truncation of the last 27 amino acids of the IL-8 Receptor A severely impaired the IL-8-induced internalization of the Receptor. Of importance was the observation that binding of IL-8 to Receptors A and B triggered a dramatically faster rate of internalization of Receptor B than Receptor A, suggesting that the heterologous C termini among Receptor subtypes modulate the rate of internalization of IL-8 Receptors. However, substitution of the C terminus of the Receptor subtype A for the C terminus of Receptor B reduced the internalization rate of Receptor A. Furthermore, we found that the rate of internalization of IL-8 Receptor B triggered by IL-8 was faster than the one induced by the IL-8-related peptide, melanoma growth stimulatory activity. Studies with human neutrophils pretreated with 100 nM IL-8 for 5 min revealed a positive and a negative calcium response mediated by Receptors A and B, respectively. In contrast, neutrophils pretreated with melanoma growth stimulatory activity showed positive calcium responses to both Receptors A and B. These data suggest that the neutrophil responses mediated by IL-8 are modulated by the rate of internalization of Receptors.

  • amino terminus of the Interleukin 8 Receptor is a major determinant of Receptor subtype specificity
    Journal of Biological Chemistry, 1992
    Co-Authors: Gregory Larosa, Kathleen M Thomas, Linda Taylor, Mark E Kaufmann, Robert Mark, Michael White, Gary S Gray, Daniel P Witt, Javier Navarro
    Abstract:

    Interleukin-8 (IL-8) is a key mediator in the migration of neutrophils from the circulation to the site of inflammation in the tissue. IL-8 is secreted by many cell types in response to proinflammatory stimuli such as Interleukin 1, tumor necrosis factor, and lipopolysaccharide and is a potent chemoattractant and activator of neutrophils. Neutrophil activating peptide-2 (NAP-2) and melanoma growth-stimulatory activity (MGSA/GRO) are structurally and functionally related to IL-8 and, like IL-8, bind to specific G protein-coupled Receptors on neutrophils. In the present study two closely related cloned IL-8 Receptor subtypes are characterized by expression of the cDNA clones in monkey kidney cells (COS-7) or chinese hamster ovary cells and analysis of their ligand binding profiles. Both Receptor subtypes bind 125I-labeled IL-8 with similar high affinity, however, the F3R Receptor binds IL-8 exclusively, while the 4Ab Receptor binds both IL-8 and MGSA/GRO with high affinity and NAP-2 with lesser affinity. Furthermore, we demonstrate with the use of intersubtype chimeric Receptors that the specificity of ligand binding to both IL-8 Receptor subtypes is dictated by the heterogeneous NH2-terminal domain. The F3R Receptor is representative of a restricted IL-8 Receptor subtype, and 4Ab represents a nonrestricted Receptor subtype. It is proposed that these subtypes be named IL-8 Receptors alpha and beta, respectively.

  • the Interleukin 8 Receptor is encoded by a neutrophil specific cdna clone f3r
    Journal of Biological Chemistry, 1991
    Co-Authors: Kathleen M Thomas, Linda Taylor, Javier Navarro
    Abstract:

    Interleukin-8 (IL-8) is one of the most potent chemotactic agents for neutrophils and has been implicated as a major mediator of inflammation. The IL-8 Receptor is expressed exclusively in neutrophils and belongs to the family of G-protein-coupled Receptors. In a recent paper we reported the characterization of a cDNA clone, F3R, isolated from a neutrophil cDNA library and showed that it encodes a G-protein-coupled Receptor which is exclusively expressed in neutrophils. We also suggested, based on expression studies in Xenopus oocytes, that the F3R protein product is an isoform of the (fMLP) Receptor (Thomas, K. M., Pyun, H. Y., and Navarro, J. (1990) J. Biol. Chem. 265, 20061-20064). In this work, the F3R Receptor cDNA is expressed in monkey kidney cells (COS-7) and is shown to encode the IL-8 Receptor. F3R cDNA does not encode for a fMLP Receptor isoform. We show conclusively that the F3R-transfected COS-7 cells express the IL-8 Receptor at a density equivalent to that observed in neutrophils. The pharmacological profile of the F3R-transfected cells is the same as that of neutrophils. The apparent Kd values for binding of 125I-IL-8 to neutrophils and F3R-transfected COS-7 cell membranes were 1.2 and 1.4 nM, respectively. Antipeptide antibodies against a partial sequence of the F3R protein product specifically immunoprecipitate the IL-8 Receptor from transfected cells as well as neutrophils. The molecular characterization of the IL-8 Receptor should provide the basis for further studies on the identification of the binding domain of this inflammatory Receptor.

Sunil K. Ahuja - One of the best experts on this subject based on the ideXlab platform.

  • the cxc chemokines growth regulated oncogene gro α groβ groγ neutrophil activating peptide 2 and epithelial cell derived neutrophil activating peptide 78 are potent agonists for the type b but not the type a human Interleukin 8 Receptor
    Journal of Biological Chemistry, 1996
    Co-Authors: Sunil K. Ahuja, Philip M Murphy
    Abstract:

    Abstract Interleukin-8 (IL-8), growth-related oncogene (GRO) α, GROβ, GROγ, neutrophil-activating peptide-2 (NAP-2), epithelial cell-derived neutrophil activating peptide- 78 (ENA-78), and granulocyte chemoattractant protein-2 are potent neutrophil chemoattractants 40-90% identical in amino acid sequence that comprise a subgroup of human CXC chemokines defined by the conserved sequence motif glutamic acid-leucine-arginine (ELR). Two human chemotactic Receptor subtypes for IL-8, named IL-8 Receptors (IL8R) A and B, have been cloned. They are 78% identical in amino acid sequence, coexpressed in neutrophils, and distinguished by their different selectivities for GROα and NAP-2. Their selectivity for other ELR+ CXC chemokines has not been previously reported. By measuring calcium flux in human embryonic kidney 293 cells transfected with plasmids encoding IL8RA or IL8RB, we have now defined Receptor selectivity for GROβ, GROγ, and ENA-78. The rank order of agonist potency, based on inspection of the mean effective concentration values (EC50), for IL8RB was GROγ (1 nM) > IL-8 (4 nM) ∼ GROα (5 nM) ∼ GROβ (4 nM) ∼ NAP-2 (7 nM) > ENA-78 (11 nM), and for IL8RA was IL-8 (4 nM) >>> ENA-78 (40 nM) ∼ NAP-2 (45 nM) > GROα (63 nM)∼GROγ (65 nM) ≫ GROβ. The maximal response of IL8RA to IL-8 was at least 2-fold greater than the other five chemokines. All six agonists for IL8RB competed for high affinity 125I-IL-8, -GROα, -NAP-2, and -ENA-78 binding sites at IL8RB. GROα, GROβ, GROγ, NAP-2, and ENA-78 competed weakly for the high affinity IL-8 binding site at IL8RA. Thus, IL8RA and IL8RB are both highly selective for IL-8 and have similar sequences but differ dramatically in their selectivity for all other ELR+ CXC chemokines tested. These findings have important implications for developing novel neutrophil-specific anti-inflammatory drugs directed against the CXC chemokine signaling system.

  • cxc chemokines bind to unique sets of selectivity determinants that can function independently and are broadly distributed on multiple domains of human Interleukin 8 Receptor b determinants of high affinity binding and Receptor activation are distinct
    Journal of Biological Chemistry, 1996
    Co-Authors: Sunil K. Ahuja, Jennifer C Lee, Philip M Murphy
    Abstract:

    Abstract Human Interleukin-8 Receptors A (IL-8RA) and B (IL-8RB) are seven-transmembrane domain (TMD) neutrophil chemokine Receptors with similar sequences (77% amino acid identity) and similar G protein selectivity, but markedly different selectivity for CXC chemokines. IL-8RB is selective for IL-8, growth-related oncogene α (GROα) and neutrophil-activating peptide-2 (NAP-2), whereas IL-8RA is selective only for IL-8. To identify selectivity determinants, we made eight chimeric Receptors exchanging: 1) the three main regions of sequence divergence between IL-8RA and IL-8RB (the N-terminal segment before TMD1, the region from TMD4 to the end of the second extracellular (e2) loop, and the C-terminal tail), and 2) the N-terminal segment of CC chemokine Receptor 1, which does not bind CXC chemokines. Chimeras were tested by direct I-IL-8, I-GROα, and I-NAP-2 binding, heterologous competition binding, and calcium flux assays using human embryonic kidney 293 cells stably transfected with Receptor DNAs. The following results were obtained: 1) chimeric Receptors had binding sites for IL-8, GROα and NAP-2 distinct from those on IL-8RA and IL-8RB; 2) IL-8, GROα and NAP-2 bound to overlapping but distinct sites that mapped differentially to multiple domains on IL-8RB; 3) high affinity radioligand binding and high agonist potency were separable functions for IL-8, GROα and NAP-2, suggesting that the determinants of high affinity binding may not be critical for Receptor activation; and 4) determinants of GROα and NAP-2 selectivity were found in both the N-terminal segment before TMD1 and the region from TMD4 to the end of the e2 loop of IL-8RB, and functioned independently of each other. Stated reciprocally, the N-terminal segment of IL-8RA was not a dominant selectivity determinant. These data suggest that both narrow and broad spectrum chemokine antagonists can be developed to block functions mediated by IL-8RB.

  • Molecular piracy of mammalian Interleukin-8 Receptor type B by herpesvirus saimiri.
    Journal of Biological Chemistry, 1993
    Co-Authors: Sunil K. Ahuja, Philip M Murphy
    Abstract:

    Abstract Viruses are known to acquire and modify the genes of their hosts to attain a survival advantage in the host environment. Herpesvirus saimiri (HVS) is a T-lymphotropic virus that causes fatal lymphoproliferative diseases in several non-human primates. The gene ECRF3 of HVS was most likely acquired from a primate host. ECRF3 encodes a putative seven-transmembrane-domain Receptor that is remotely related (approximately 30% amino acid identity) to the known mammalian alpha and beta chemokine Receptors, namely Interleukin-8 Receptor (IL8R) types A and B and the MIP-1 alpha/RANTES Receptor, respectively. Chemokines regulate the trafficking, activation, and, in some cases, proliferation of myeloid and lymphoid cell types. We now show that ECRF3 encodes a functional Receptor for the alpha chemokines IL-8, GRO/melanoma growth stimulatory activity (MGSA), and NAP-2 but not for beta chemokines, a specificity identical to that of IL8RB. Paradoxically, IL8RA shares 77% amino acid identity with IL8RB but is not a Receptor for GRO/MGSA or NAP-2. This is the first functional characterization of a viral seven-transmembrane-domain Receptor. It suggests a novel role for alpha chemokines in the pathogenesis of HVS infection by transmembrane signaling via the product of ECRF3.

  • molecular evolution of the human Interleukin 8 Receptor gene cluster
    Nature Genetics, 1992
    Co-Authors: Sunil K. Ahuja, Tayfun Ozcelik, Athena Milatovitch, Uta Francke, Philip M Murphy
    Abstract:

    lnterleukin–8 (IL–8) is the prototype for a family of at least eight neutrophil chemoattractants whose genes map to human chromosome 4q13–q21. Two human IL–8 Receptors, IL8RA and IL8RB, are known from cDNA cloning; IL8RA is a promiscuous Receptor for at least two other related ligands, GROα and NAP–2. We now report cloning of the genes for IL8RA, IL8RB and a recently inactivated pseudogene of Receptor A (IL8RAP). These form a cluster of only three genes in the superfamily of G protein–coupled Receptors (GPCRs) and map to 2q34–q35. The revolutionary diversity displayed by the IL–8 ligand–Receptor complex — ligand promiscuity for IL–8, Receptor promiscuity for IL8RA, gene duplication for both ligands and Receptors and gene extinction in the case of IL8RAP — is unprecedented for the GPCR superfamily.

Norma P Gerard - One of the best experts on this subject based on the ideXlab platform.

  • cloning of the human c5a anaphylatoxin Receptor and more
    Frontiers in Immunology, 2015
    Co-Authors: Norma P Gerard, Craig Gerard
    Abstract:

    The initial observation in 1973 that the complement C5 activation product, C5a, has the ability to stimulate human neutrophils led to the concept of a specific C5a Receptor (1). In the mid-1986, a groundbreaking paper published in Nature announced the cloning of the beta-adrenergic Receptor, and for the first time established seven transmembrane (7TM) G protein-coupled Receptors as members of the rhodopsin superfamily (2). Almost simultaneously, Feltner and colleagues demonstrated that the FMLP, C5a, and LTB4 activities on rabbit neutrophils could be inhibited by pertussis toxin, indicating coupling to GTP binding proteins (3). In 1987, Masu and colleagues used an oocyte expression cloning system to isolate a cDNA encoding the neuropeptide substance K Receptor; and later, the same group identified the related substance P Receptor (4, 5). In 1989, we realized that the pertussis sensitivity of the fMLP, C5a, and LTB4 Receptors suggested that they would also be members of the rhodopsin superfamily. When we aligned the handful of structures for the then known 7TM Receptors (adrenergic, serotonin, dopamine, FSH/LH, and substance P and K Receptors), we recognized homologies in both the transmembrane segments and intracellular loops, which presumably facilitated interactions with G proteins. This observation actually presented us with an opportunity to attempt to clone Receptors by homology to the superfamily as “orphan Receptors.” We constructed an antisense oligonucleotide with minimal degeneracy that encompassed a highly conserved NPXXY motif in the seventh transmembrane segment of the known rhodopsin family members. In order to enrich in C5a Receptors, we took advantage of the fact that the Receptors were induced by cyclic-AMP in U937 cells, and in retinoic acid differentiated human HL60 cells. By summer of 1990, we had isolated ~20 cDNAs using this approach from the cAMP induced U937 cell library. About half of these clones were an identical cDNA that we named NPIIY-18. Using this as a probe, we demonstrated that NPIIY-18 recognized a ~2.2 kb mRNA only in cAMP differentiated U937 cells. Northern blot analyses showed that NPIIY-18 was present only in cells known to express the C5a Receptor. As NPIIY-18 was not a full-length cDNA, we then probed the retinoic acid differentiated HL60 cell library. We isolated a full-length DNA from this library that encoded a 7TM Receptor with 25% homology to the substance K Receptor and 35% homology to the human fMLP Receptor (FPR1), which was cloned by Francois Boulay in May 1990 (6). When expressed in COS cells, we showed that NPIIY-18 encoded a high-affinity Receptor for human C5a (Figure ​(Figure1)1) (7). This work was accepted for publication in Nature in December 1990. In the summary paragraph of this manuscript, we pointed out that our approach should be helpful to clone the Receptors for the leukotrienes, platelet activating factor, Interleukin-8, and adenosine Receptors as these are all present on cAMP differentiated U937 cells. Almost simultaneously, Francois Boulay confirmed our identification of the human C5aR, which his group accomplished by expression cloning of differentiated HL-60 cells (8). His work was published in March 1991, some 4 months after ours. Figure 1 Final steps in cloning and identifying the human C5a Receptor. (A) Tertiary screen of clone NPIIY-18 using 32P-labeled oligonucleotide probe. (B) NPIIY-18 was transfected into COS7 cells and tested for binding of 125I-C5a under competitive binding conditions. ... In November 1990, within months of the identification of the human FPR1, Thomas et al. reported the cloning of the rabbit Receptor for fMLP, F3R (9), which had almost no significant homology to the human Receptor. This was troubling, because we knew that when the ligand was identical from species to species, the Receptors were generally highly conserved. Thus, the adrenergic, dopamine, serotonin, and histamine Receptors are >90% identical across species. Curiously, the cDNA was reported to bind the FPR1 radioligand and transduce calcium transients. Because the expression of the claimed rabbit F3R formyl peptide Receptor was restricted to neutrophils, we wondered if, in fact, the Navarro lab had misidentified an Interleukin-8 Receptor. One of us (Craig Gerard) actually traveled to the Navarro lab to obtain the F3R cDNA to establish a collaboration and test its identity as a Receptor for IL-8. At that time, there was no radioligand IL-8 commercially available. Henry Showell, of Pfizer Central Research, was able to provide us with a custom iodinated IL-8, which we demonstrated to bind F3R. Unfortunately, we did not have sufficient quantities of the reagent to perform comprehensive studies to publish our findings. We disclosed our result to Javier Navarro, but were left in silence. Unbeknownst to us, Dan Witt, at Repligen, had reportedly approached the Navarro lab with a similar idea. Thomas et al. went on to publish F3R as an IL8 Receptor, without retracting the previous paper (10). During this time, Tom Schall and I met at a FASEB meeting with Phil Murphy, and suggested to him that he use F3R to clone a human homolog from HL60 cells and test it against IL8. The landmark Murphy and Tiffany paper resulted (11). Phil offered one of us (Craig Gerard) coauthorship for the helpful suggestion but because of intellectual property concerns at our institution, we requested an acknowledgment instead. Over the next decade, the orphan Receptor approach led to the identification of a wide variety of chemoattractant Receptors, including most of the chemokine Receptor system. The most notable events in the area of chemokines occurred when CXCR4 and CCR5 were identified as HIV coReceptors. It was known from the work of Ed Berger that CXCR4 was the obligate coReceptor with CD4 for laboratory-adapted strains of HIV (12). However, the wild type, the so-called macrophage tropic strain used an unknown coReceptor. In December 1995, it was reported in the New York Times that the Gallo laboratory had identified Mip1α, Mip1β, and RANTES as substances that inhibited HIV infections (13). Coincidentally, at the Fourth International Chemokine Symposium, held June 27–30, 1995, Izzy Charo described an orphan Receptor identified as CCR5, linked to CCR2, which bound Mip1α, Mip1β, and RANTES (14). Thus, an international race began as five chemokine labs partnered with HIV labs to prove the hypothesis that CCR5 was the HIV coReceptor.

  • molecular characterization of the Interleukin 8 Receptor
    Biochemical and Biophysical Research Communications, 1991
    Co-Authors: Patricia M Beckmann, Carl J Kozlosky, William E Munger, Tim Vandenbos, Virginia L Price, Stewart D Lyman, Norma P Gerard, Craig Gerard, Douglas P Cerretti
    Abstract:

    Recently a rabbit cDNA (F3R) was characterized as binding and causing calcium mobilization induced by the formyl-methionine-leucine-phenylalanine peptide (fMLP). In the study reported here, cloned DNAs were isolated from rabbit genomic DNA by PCR based on the sequence of F3R. The cloned DNAs have several differences in the DNA sequence compared to the reported F3R sequence that alter the predicted protein sequence. COS-7 cells transfected with these clones in a mammalian expression vector bind human IL-8 with high affinity, but do not bind fMLP. We therefore believe that the cDNAs isolated encode the rabbit IL-8 Receptor.

Douglas P Cerretti - One of the best experts on this subject based on the ideXlab platform.

  • assignment of the genes encoding human Interleukin 8 Receptor types 1 and 2 and an Interleukin 8 Receptor pseudogene to chromosome 2q35
    Genomics, 1992
    Co-Authors: Stephan W Morris, Nicole Nelson, Marcus B Valentine, David N Shapiro, A T Look, Carl J Kozlosky, M P Beckmann, Douglas P Cerretti
    Abstract:

    Two human cDNA clones that encode different Interleukin-8 (IL8) Receptors have recently been isolated. The Interleukin-8 Receptor type 1 (IL8R1) binds IL8 only, whereas the Interleukin-8 Receptor type 2 (IL8R2) (previously designated IL8RA) also binds growth regulated gene (GRO), and neutrophil activating protein-2 (NAP-2) with high affinity. In the process of screening a genomic library with these cDNAs to obtain large clones for use in chromosomal localization studies, we isolated an Interleukin-8 Receptor pseudogene (IL8RP) that bears greatest similarity to IL8R2. Using Southern hybridization analysis of human x rodent somatic cell hybrid DNAs with cDNA probes for IL8R1 and IL8R2 and probes from the IL8RP locus, we assigned the three loci to chromosome 2; fluorescence in situ hybridization (FISH) to metaphase chromosome preparations using genomic clones from each locus refined this localization to chromosome 2, band q35, for all three. By virtue of their chromosomal location, IL8R1 and IL8R2 may be considered candidate genes for several human disorders in which the involved locus has been mapped to distal 2q or that are associated with structural abnormalities of this segment, including van der Woude syndrome and the neoplastic diseases rhabdomyosarcoma and uterine leiomyomata. In addition, because this region of chromosome 2q is homologous to proximal mouse chromosome 1 in the segment containing the Lsh-Ity-Bcg locus involved in mediating host resistance to infection with intracellular pathogens, examination for abnormalities of the murine homologues of the IL8R genes should be considered in mice affected by mutations of this locus.

  • molecular characterization of the Interleukin 8 Receptor
    Biochemical and Biophysical Research Communications, 1991
    Co-Authors: Patricia M Beckmann, Carl J Kozlosky, William E Munger, Tim Vandenbos, Virginia L Price, Stewart D Lyman, Norma P Gerard, Craig Gerard, Douglas P Cerretti
    Abstract:

    Recently a rabbit cDNA (F3R) was characterized as binding and causing calcium mobilization induced by the formyl-methionine-leucine-phenylalanine peptide (fMLP). In the study reported here, cloned DNAs were isolated from rabbit genomic DNA by PCR based on the sequence of F3R. The cloned DNAs have several differences in the DNA sequence compared to the reported F3R sequence that alter the predicted protein sequence. COS-7 cells transfected with these clones in a mammalian expression vector bind human IL-8 with high affinity, but do not bind fMLP. We therefore believe that the cDNAs isolated encode the rabbit IL-8 Receptor.