RESUMO
Chemokines mediate their diverse activities through G protein-coupled receptors. The human homolog of the bovine orphan receptor PPR1 shares significant similarity to chemokine receptors. Transfection of this receptor into murine L1.2 cells resulted in responsiveness to monocyte chemoattractant protein (MCP)-4, MCP-2, and MCP-1 in chemotaxis assays. Binding studies with radiolabeled MCP-4 demonstrated a single high affinity binding site with an IC(50) of 0.14 nM. As shown by competition binding, other members of the MCP family also recognized this receptor. MCP-2 was the next most potent ligand, with an IC(50) of 0.45 nM. Surprisingly, eotaxin (IC(50) = 6.7 nM) and MCP-3 (IC(50) = 4.1 nM) bind with greater affinity than MCP-1 (IC(50) = 10.7 nM) but only act as agonists in chemotaxis assays at 100-fold higher concentrations. Because of high affinity binding and functional chemotactic responses, we have termed this receptor CCR11. The gene for CCR11 was localized to human chromosome 3q22, which is distinct from most CC chemokine receptor genes at 3p21. Northern blot hybridization was used to identify CCR11 expression in heart, small intestine, and lung. Thus CCR11 shares functional similarity to CCR2 because it recognizes members of the MCP family, but CCR11 has a distinct expression pattern.
Assuntos
Proteínas Quimioatraentes de Monócitos/metabolismo , Receptores de Quimiocinas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Mapeamento Cromossômico , Cromossomos Humanos Par 3 , DNA Complementar , Humanos , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Receptores CCR , Receptores de Quimiocinas/química , Receptores de Quimiocinas/genética , Homologia de Sequência de AminoácidosRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 3 genes that are homologous to cellular chemokines. vMIP-III, the product of open reading frame K4.1, is the most distantly related to human chemokines and has yet to be characterized. We have examined the interaction of vMIP-III with chemokine receptors, its expression in KS lesions, and its in ovo angiogenic properties. We show expression of vMIP-III in KS lesions and demonstrate the stimulation of angiogenesis by this chemokine, like vMIP-I and vMIP-II, in the chick chorioallantoic membrane assay. vMIP-III does not block human immunodeficiency virus entry through the coreceptors CCR3, CCR5, or CXCR4. However, vMIP-III is an agonist for the cellular chemokine receptor CCR4. CCR4 is expressed by TH2-type T cells. Consistent with this, vMIP-III preferentially chemoattracts this cell type. Because of these biologic properties and because it is expressed in KS lesions, vMIP-III may play an important role in the pathobiology of KS. (Blood. 2000;95:1151-1157)
Assuntos
Quimiocinas CC/genética , Quimiocinas CC/farmacologia , Quimiocinas CC/fisiologia , Herpesvirus Humano 8/genética , Neovascularização Fisiológica/fisiologia , Receptores de Quimiocinas/agonistas , Células Th2/fisiologia , Alantoide/irrigação sanguínea , Animais , Células CHO , Linhagem Celular , Quimiocinas CC/imunologia , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , Embrião de Galinha , Córion/irrigação sanguínea , Cricetinae , HIV/efeitos dos fármacos , HIV/fisiologia , Herpesvirus Humano 8/imunologia , Humanos , Neovascularização Fisiológica/efeitos dos fármacos , Fases de Leitura Aberta , Receptores CCR4 , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Sarcoma de Kaposi/imunologia , Sarcoma de Kaposi/patologia , Sarcoma de Kaposi/virologia , Células Th1/efeitos dos fármacos , Células Th1/fisiologia , Células Th2/efeitos dos fármacos , Transfecção , Proteínas Virais , Replicação Viral/efeitos dos fármacosRESUMO
Human (H-) CCR5 is the primary coreceptor for ENV-mediated fusion by R5 strains of human immunodeficiency virus type 1, whereas mouse (M-) CCR5 lacks this function. An array of 23 H/M-CCR5 hybrids containing increasing amounts of H-CCR5 extending from the N terminus generated by random chimeragenesis had a biphasic pattern of coreceptor activity with JRFL and 89.6, revealing active regions in the N-terminal extracellular domain (N-ED) and at the junction of cytoplasmic loop 3. The M-CCR5 mutant in which divergent residues were replaced with the corresponding H-CCR5 N-ED sequence (NyYTsE) gained coreceptor function in fusion but not infection experiments. A M-CCR5 double mutant with substitution of human sequences for divergent residues from the N-ED and cytoplasmic loop 3 had augmented coreceptor activity in fusion assays and gain of function in infection experiments. The SIV-251 ENV utilized H- and M-CCR5 and variants. Flow cytometric analysis of M-CCR5 mutants and bifunctional receptors composed of CD4 domains fused to M-CCR5 mutants excluded the possibility that differences in coreceptor activity resulted from variations in cell surface expression. These results demonstrate that the coreceptor activity of the H-CCR5 N-ED is modulated by intracellular residues, illustrating the complexity of CCR5 requirements for interaction with ENV.
Assuntos
HIV-1/metabolismo , Receptores CCR5/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Receptores CCR5/química , Receptores CCR5/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
High throughput partial sequencing of randomly selected cDNA clones has proven to be a powerful tool for examining the relative abundance of mRNAs and for the identification of novel gene products. Because of the important role played by macrophages in immune and inflammatory responses, we sequenced over 3000 randomly selected cDNA clones from a human macrophage library. These sequences represent a molecular inventory of mRNAs from macrophages and provide a catalog of highly expressed transcripts. Two of the most abundant clones encode recently identified CC chemokines. Macrophage-derived chemokine (MDC) plays a complex role in immunoregulation and is a potent chemoattractant for dendritic cells, T cells, and natural killer cells. The chemokine receptor CCR4 binds MDC with high affinity and also responds by calcium flux and chemotaxis. CCR4 has been shown to be expressed by Th2 type T cells. Recent studies also implicate MDC as a major component of the host defense against human immunodeficiency virus.
Assuntos
Quimiocinas/biossíntese , Quimiocinas/genética , DNA Complementar/análise , Macrófagos/metabolismo , RNA Mensageiro/metabolismo , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , HumanosRESUMO
Unique among known human herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) encodes chemokine-like proteins (vMIP-I and vMIP-II). vMIP-II was shown to block infection of human immunodeficiency virus-type 1 (HIV-1) on a CD4-positive cell line expressing CCR3 and to a lesser extent on one expressing CCR5, whereas both vMIP-I and vMIP-II partially inhibited HIV infection of peripheral blood mononuclear cells. Like eotaxin, vMIP-II activated and chemoattracted human eosinophils by way of CCR3. vMIP-I and vMIP-II, but not cellular MIP-1alpha or RANTES, were highly angiogenic in the chorioallantoic assay, suggesting a possible pathogenic role in Kaposi's sarcoma.
Assuntos
Quimiocinas/fisiologia , HIV-1/fisiologia , Herpesvirus Humano 8/genética , Proteínas Inflamatórias de Macrófagos/fisiologia , Neovascularização Patológica/etiologia , Receptores de Quimiocinas , Proteínas Virais , Animais , Linfócitos T CD4-Positivos/virologia , Quimiocinas/genética , Quimiocinas/metabolismo , Quimiocinas/farmacologia , Quimiotaxia de Leucócito , Embrião de Galinha , Eosinófilos/fisiologia , Herpesvirus Humano 8/fisiologia , Humanos , Leucócitos Mononucleares/virologia , Proteínas Inflamatórias de Macrófagos/genética , Proteínas Inflamatórias de Macrófagos/metabolismo , Proteínas Inflamatórias de Macrófagos/farmacologia , Neutrófilos/fisiologia , Receptores CCR3 , Receptores de Citocinas/agonistas , Receptores de Citocinas/metabolismo , Receptores de HIV/metabolismo , Células Tumorais Cultivadas , Replicação ViralRESUMO
CCR5 and CXCR4 are the two major coreceptors that have been identified for human immunodeficiency virus (HIV) entry. We have modified several beta-galactosidase-based HIV indicator cell lines to express CCR5 and/or CXCR4. Using these new reagents, we have been able to detect all primary isolates tested using one or both of these cell lines. However, there is large variation in the absolute viral infectivity among primary strains. Furthermore, all HIV strains are capable of causing syncytia in the indicator cells when the coreceptor is present regardless of whether they had previously been characterized as "syncytia-inducing" or "non-syncytium-inducing."
Assuntos
HIV-1/isolamento & purificação , Proteínas de Membrana/metabolismo , Receptores de Citocinas/metabolismo , Receptores de HIV/metabolismo , Vírus da Imunodeficiência Símia/isolamento & purificação , Animais , Linhagem Celular , Células Gigantes , HIV-1/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/genética , Receptores CCR5 , Receptores CXCR4 , Receptores de Citocinas/genética , Receptores de HIV/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vírus da Imunodeficiência Símia/metabolismoRESUMO
Chemokines constitute a family of low-molecular-weight proteins that attract or activate a variety of cell types, including leukocytes, endothelial cells, and fibroblasts. An electronic search of the GenBank Expressed Sequence Tags database uncovered a partial cDNA sequence with homology to the chemokine monocyte chemotactic protein-1 (MCP-1). Isolation of the full-length clone revealed that it encodes the chemokine MCP-4, an eosinophil chemoattractant recently described by Uguccioni et al. [J. Exp. Med. 183, 2379-2384]. Recombinant MCP-4 was expressed in mammalian cells and purified by heparin-Sepharose chromatography. Sequencing the amino terminus of this protein corroborated the reported sequence of recombinant MCP-4 produced in insect cells. As shown by calcium flux assays, MCP-4 activated the cloned G protein-coupled receptor CCR-2, which also recognizes MCP-1 and MCP-3. Northern hybridization indicated that MCP-4 is constitutively expressed at high levels in the small intestine, colon, and lung. This expression profile is consistent with its role as a chemoattractant for eosinophils, which can be rapidly mobilized to the lung or intestine in response to invading pathogens. In marked contrast to MCP-1, MCP-4 was not induced in cell lines treated with pro-inflammatory stimuli such as lipopolysaccharide or tumor necrosis factor alpha.
Assuntos
Cálcio/metabolismo , Citocinas , DNA Complementar/genética , Proteínas Quimioatraentes de Monócitos/genética , Receptores de Quimiocinas , Receptores de Citocinas/efeitos dos fármacos , Animais , Sequência de Bases , Células CHO , Linhagem Celular , Quimiocina CCL7 , Cricetinae , DNA Complementar/isolamento & purificação , Humanos , Dados de Sequência Molecular , Proteínas Quimioatraentes de Monócitos/metabolismo , Proteínas Quimioatraentes de Monócitos/farmacologia , RNA Mensageiro/metabolismo , Receptores CCR2 , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , TransfecçãoRESUMO
Chemokines affect leukocyte chemotactic and activation activities through specific G protein-coupled receptors. In an effort to map the closely linked CC chemokine receptor genes, we identified a novel chemokine receptor encoded 18 kilobase pairs downstream of the monocyte chemoattractant protein-1 (MCP-1) receptor (CCR2) gene on human chromosome 3p21. The deduced amino acid sequence of this novel receptor, designated CCR5, is most similar to CCR2B, sharing 71% identical residues. Transfected cells expressing the receptor bind RANTES (regulated on activation normal T cell expressed), MIP-1beta, and MIP-1alpha with high affinity and generate inositol phosphates in response to these chemokines. This same combination of chemokines has recently been shown to potently inhibit human immunodeficiency virus replication in human peripheral blood leukocytes (Cocchi, F., DeVico, A. L., Garzino-Demo, A., Arya, S. K., Gallo, R. C., and Lusso, P.(1995) Science 270, 1811-1815). CCR5 is expressed in lymphoid organs such as thymus and spleen, as well as in peripheral blood leukocytes, including macrophages and T cells, and is the first example of a human chemokine receptor that signals in response to MIP-1beta.
Assuntos
Quimiocina CCL5/metabolismo , Cromossomos Humanos Par 3 , Monocinas/metabolismo , Receptores de Citocinas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Quimiocina CCL3 , Quimiocina CCL4 , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Clonagem Molecular , Primers do DNA , Feminino , Biblioteca Gênica , Inibidores do Crescimento/metabolismo , Humanos , Cinética , Proteínas Inflamatórias de Macrófagos , Masculino , Dados de Sequência Molecular , Família Multigênica , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Receptores CCR5 , Receptores de Citocinas/química , Receptores de Citocinas/genética , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
Chemokines are relatively small peptides with potent chemoattractant and activation activities for leukocytes. Several chemokine receptors have been cloned and characterized and all are members of the G protein-coupled receptor superfamily. Using degenerate oligonucleotides and polymerase chain reaction, we have identified seven novel receptors. Two of these sequences are presented here for the first time. We have shown, with gene mapping studies, that receptors with the highest sequence similarity are closely linked on human chromosomes. This close genetic association suggests a functional relationship as well.
Assuntos
Quimiocinas/genética , Quimiocinas/metabolismo , Receptores de Citocinas/genética , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Receptores de Citocinas/fisiologiaRESUMO
A polymerase chain reaction (PCR) strategy with degenerate primers was used to identify novel G-protein-coupled receptor-encoding genes from human genomic DNA. One of the isolated clones, termed V28, showed high sequence similarity to the genes encoding human chemokine receptors for monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1 alpha (MIP-1 alpha)/RANTES, and to the rat orphan receptor-encoding gene RBS11. When RNA was analyzed by Northern blot, V28 was found to be most highly expressed in neural and lymphoid tissues. Myeloid cell lines, particularly THP.1 cells, showed especially high expression of V28. We have mapped V28 to human chromosome 3p21-3pter, near the MIP-1 alpha/RANTES receptor-encoding gene.
Assuntos
Quimiocina CCL2/genética , Citocinas/genética , Monocinas/genética , Receptores de Superfície Celular/genética , Receptores de Citocinas/genética , Receptores de HIV/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Receptor 1 de Quimiocina CX3C , Quimiocina CCL3 , Quimiocina CCL4 , Mapeamento Cromossômico , Cromossomos Humanos Par 3 , Proteínas de Ligação ao GTP/metabolismo , Humanos , Tecido Linfoide/metabolismo , Proteínas Inflamatórias de Macrófagos , Dados de Sequência Molecular , Neurônios/metabolismo , Ratos , Receptores de Superfície Celular/biossínteseRESUMO
A lymphoid-specific member of the G-protein-coupled receptor family has been identified by PCR with degenerate oligonucleotides. We have determined that this receptor, also reported as the Epstein-Barr-induced cDNA EBI1, is expressed in normal lymphoid tissues and in several B- and T-lymphocyte cell lines. While the function and the ligand for EBI1 remain unknown, its sequence and gene structure suggest that it is related to the receptors that recognize chemoattractants, such as interleukin-8, RANTES, C5a, and fMet-Leu-Phe. Like the chemoattractant receptors, EBI1 contains intervening sequences near its 5' end; however, EBI1 is unique in that both of its introns interrupt the coding region of the first extracellular domain. The gene is encoded on human chromosome 17q12-q21.2. None of the other G-protein-coupled receptors has been mapped to this region, but the C-C chemokine family has been mapped to 17q11-q21. The mouse EBI1 cDNA has also been isolated and encodes a protein with 86% identity to the human homolog.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 17 , Proteínas de Ligação ao GTP/genética , Hominidae/genética , Camundongos/genética , Receptores de Superfície Celular/genética , Receptores de Quimiocinas , Sequência de Aminoácidos , Animais , Linfócitos B/metabolismo , Sequência de Bases , Clonagem Molecular , Primers do DNA , Éxons , Biblioteca Genômica , Herpesvirus Humano 4/genética , Humanos , Íntrons , Leucócitos/metabolismo , Linfócitos/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Receptores CCR7 , Receptores de Superfície Celular/biossíntese , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Linfócitos T/metabolismoRESUMO
Platelet-activating factor (PAF), a phospholipid, exhibits a variety of potent inflammatory bioactivities that are mediated by a specific cell surface receptor. The gene for the human PAF receptor (PTAFR) has been isolated by hybridization with a guinea pig probe. The coding sequence contains no intervening sequences. The encoded protein is highly homologous to the guinea pig PAF receptor (82% identity) and contains seven putative transmembrane domains. The PAF receptor therefore appears to be a member of the G protein coupled family of receptors and exhibits significant similarity to many members of the family. Analysis of somatic cell hybrids suggests that the PAF receptor is encoded by a single gene on human chromosome 1.
Assuntos
Cromossomos Humanos Par 1 , Fator de Ativação de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , DNA/genética , Sondas de DNA , Humanos , Íntrons , Dados de Sequência MolecularRESUMO
Complementary DNA (cDNA) clones encoding human macrophage-specific specific colony-stimulating factor (CSF-1) were isolated. One cDNA clone codes for a mature polypeptide of 224 amino acids and a putative leader of 32 amino acids. This cDNA, which was cloned in the Okayama-Berg expression vector, specifies the synthesis of biologically active CSF-1 in COS cells, as determined by a specific radioreceptor assay, macrophage bone marrow colony formation, and antibody neutralization. Most of the cDNA isolates contain part of an intron sequence that changes the reading frame, resulting in an abrupt termination of translation; these cDNA's were inactive in COS cells. The CSF-1 appears to be encoded by a single-copy gene, but its expression results in the synthesis of several messenger RNA species, ranging in size from about 1.5 to 4.5 kilobases.
Assuntos
Clonagem Molecular , Fatores Estimuladores de Colônias/genética , DNA/metabolismo , Genes , Macrófagos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Enzimas de Restrição do DNA , Humanos , Neoplasias Pancreáticas , RNA Mensageiro/genética , Transcrição GênicaRESUMO
The filamentous ascomycete Aspergillus awamori secretes large amounts of glucoamylase upon growth in medium containing starch, glucose, or a variety of hexose sugars and sugar polymers. We examined the mechanism of this carbon source-dependent regulation of glucoamylase accumulation and found a several hundredfold increase in glucoamylase mRNA in cells grown on an inducing substrate, starch, relative to cells grown on a noninducing substrate, xylose. We postulate that induction of glucoamylase synthesis is regulated transcriptionally. Comparing total mRNA from cells grown on starch and xylose, we were able to identify an inducible 2.3-kilobase mRNA-encoding glucoamylase. The glucoamylase mRNA was purified and used to identify a molecularly cloned 3.4-kilobase EcoRI fragment containing the A. awamori glucoamylase gene. Comparison of the nucleotide sequence of the 3.4-kilobase EcoRI fragment with that of the glucoamylase I mRNA (as determined from molecularly cloned cDNA) revealed the existence of four intervening sequences within the glucoamylase gene. The 5' end of the glucoamylase mRNA was mapped to several locations within a region -52 to -73 nucleotides from the translational start. Sequence and structural features of the glucoamylase gene of the filamentous ascomycete A. awamori were examined and compared with those reported in genes of other eucaryotes.
Assuntos
Aspergillus/genética , Genes Fúngicos , Glucana 1,4-alfa-Glucosidase/genética , Glucosidases/genética , Sequência de Bases , Clonagem Molecular , Regulação da Expressão Gênica , RNA Fúngico/genética , RNA Mensageiro/genéticaRESUMO
Two new cloning vectors (pBEU28 and pBEU50) with temperature-controlled runaway-replication properties are described. pBEU28 is similar to aphA+ (KanR) plasmid pBEU2 but lacks a 1.8-kb duplication which is responsible for plasmid instability. pBEU50 is an analog of pBR313 and pBR322 in that it carries bla+(AmpR), which can be used for selection, and tet+(TetR) which can be inactivated by cloning at HindIII and BamHI restriction sites. Sublethal concentrations of novobiocin were exploited to suppress runaway replication and to restore the viability of the plasmid carriers. By this method copB deletion mutants of two temperature-controlled, conditional runaway-replication plasmids were detected and isolated. The unconditional runaway-replication property of these plasmids leads us to hypothesize that there are at least two controls of plasmid R1 copy number and that the copB-dependent control is temperature-sensitive in the conditional runaway replication mutants. The novobiocin suppression of the runaway replication permitted us to clone dnaN+ on pBEU28 and to identify its presence at 42 degrees C with a dnaN59 transformation recipient which was temperature-sensitive due to a defect in the dnaN gene.