RESUMEN

The negative regulation of T- or B-cell antigen receptor signaling by CD5 was proposed based on studies of thymocytes and peritoneal B-1a cells from CD5-deficient mice. Here, we show that CD5 is constitutively associated with phosphotyrosine phosphatase activity in Jurkat T cells. CD5 was found associated with the Src homology 2 (SH2) domain containing hematopoietic phosphotyrosine phosphatase SHP-1 in both Jurkat cells and normal phytohemagglutinin-expanded T lymphoblasts. This interaction was increased upon T-cell receptor (TCR)-CD3 cell stimulation. CD5 co-cross-linking with the TCR-CD3 complex down-regulated the TCR-CD3-increased Ca2+ mobilization in Jurkat cells. In addition, stimulation of Jurkat cells or normal phytohemagglutinin-expanded T lymphoblasts through TCR-CD3 induced rapid tyrosine phosphorylation of several protein substrates, which was substantially diminished after CD5 cross-linking. The CD5-regulated substrates included CD3zeta, ZAP-70, Syk, and phospholipase Cgammal but not the Src family tyrosine kinase p56(lck). By mutation of all four CD5 intracellular tyrosine residues to phenylalanine, we found the membrane-proximal tyrosine at position 378, which is located in an immunoreceptor tyrosine-based inhibitory (ITIM)-like motif, crucial for SHP-1 association. The F378 point mutation ablated both SHP-1 binding and the down-regulating activity of CD5 during TCR-CD3 stimulation. These results suggest a critical role of the CD5 ITIM-like motif, which by binding to SHP-1 mediates the down-regulatory activity of this receptor.

Asunto(s)

Antígenos CD5/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Dominios Homologos src , Antígenos CD , Antígenos de Diferenciación de Linfocitos T , Sitios de Unión , Complejo CD3/metabolismo , Calcio/metabolismo , Precursores Enzimáticos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Isoenzimas/metabolismo , Células Jurkat , Fosfolipasa C gamma , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Quinasas/metabolismo , Agregación de Receptores , Proteínas Tirosina Fosfatasas con Dominio SH2 , Transducción de Señal , Quinasa Syk , Fosfolipasas de Tipo C/metabolismo , Proteína Tirosina Quinasa ZAP-70

RESUMEN

CD6 is a cell surface glycoprotein that functions both as a co-stimulatory and adhesion receptor on T cells. Recently we have described CD6 isoforms (CD6a, b, c, d, e) that arise via alternative splicing of exons encoding the cytoplasmic region of the molecule. CD6 becomes phosphorylated on tyrosine (Tyr) residues following stimulation through the T cell receptor (TCR) complex. Since the phosphorylation of Tyr residues renders some cell surface receptors competent for interactions with proteins of intracellular signaling pathways, we wanted to determine which region(s) and residues in the cytoplasmic domain of CD6 were important for phosphorylation on Tyr residues. We engineered and stably expressed chimeric receptors that consisted of the extracellular region of mouse CD6 and the cytoplasmic regions of either naturally occurring isoforms of human CD6, truncated proteins, or point mutants. We were able to demonstrate that of the nine Tyr residues in the cytoplasmic domain of the largest isoform CD6a, the two C-terminal Tyr residues (Tyr 629/662) are critical for the phosphorylation of CD6 following TCR cross-linking. Isoform CD6e, which is missing a region that contains two proline-rich motifs, is not phosphorylated. We further analyzed the ability of the different CD6 isoforms and truncated receptors to mobilize intracellular calcium after CD6/TCR co-ligation. All CD6 isoforms, including CD6e, as well as the truncation mutant delta 555, which is missing approximately the C-terminal half of the cytoplasmic domain, are able to increase Ca2+ influx. Taken together, these results suggest that the region of CD6 which is critical for Ca2+ mobilization is located N-terminal from amino acid 555 and is therefore different from the region located at the C terminus of CD6, which is necessary for tyrosine phosphorylation.

Asunto(s)

Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Calcio/metabolismo , Citoplasma/inmunología , Citoplasma/metabolismo , Tirosina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Antígenos CD/química , Antígenos CD/genética , Antígenos de Diferenciación de Linfocitos T/química , Antígenos de Diferenciación de Linfocitos T/genética , Técnicas de Cultivo de Célula , Citoplasma/química , Humanos , Isomerismo , Células Jurkat , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Mutación Puntual , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Tirosina/genética

RESUMEN

Activated leukocyte cell adhesion molecule (ALCAM; CD166) is a member of the immunoglobulin gene superfamily (IgSF) which is expressed by activated leukocytes and thymic epithelial cells and is a ligand for the lymphocyte antigen CD6. Herein, we report on the isolation and characterization of cDNA clones encoding mouse ALCAM (mALCAM). Comparison of the predicted amino acid sequence of mALCAM and human ALCAM (hALCAM) showed an overall identity of 93%. Binding studies with truncated forms of the extracellular region of mALCAM showed that the CD6 binding site is located in the N-terminal Ig-like domain and that mALCAM is capable of binding both human and mouse CD6. Mutagenesis studies on hALCAM suggested that residues critical for CD6 binding map to the predicted A'GFCC'C" beta-sheet of ALCAM's N-terminal binding domain. Residue differences in the N-terminal domains of mALCAM and hALCAM were analyzed with the aid of a molecular model of ALCAM. All residues critical for CD6 binding are conserved in both mALCAM and hALCAM, whereas residue differences map to the predicted BED face which is opposite the CD6 binding site on hALCAM. These findings provide a molecular rationale for the observed cross-species CD6/ALCAM interaction and the apparent inability to generate monoclonal antibodies (mAb) against the CD6 binding site. RNA blot analysis showed that mRNA transcripts encoding mALCAM are expressed in the brain, lung, liver, and the kidney, as well as by activated leukocytes and a number of cell lines. A rat mAb specific for mALCAM was produced and by two-color immunofluorescence studies was shown to bind to both activated CD4+ and CD8+ T cells.

Asunto(s)

Antígenos CD/metabolismo , Moléculas de Adhesión Celular/química , Glicoproteínas/química , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Molécula de Adhesión Celular del Leucocito Activado , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , Antígenos CD/inmunología , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/metabolismo , Clonación Molecular , Secuencia Conservada , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Humanos , Células L , Ligandos , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos/inmunología , Unión Proteica/inmunología , Ratas , Especificidad de la Especie , Timo , Células Tumorales Cultivadas

RESUMEN

The CD6 protein has been shown to play important roles in T cell costimulation and adhesion. Recently, variably spliced isoforms of CD6 mRNA have been identified in both human and murine T cells. Here we report on the genomic organization of the human CD6 gene, its chromosomal localization, and the characterization of novel isoforms. Human CD6 is encoded by at least 13 exons. The amino terminal signal sequence, extracellular region, and transmembrane domain are encoded by seven exons, while the cytoplasmic domain of CD6 is encoded by six exons. Each of the three extracellular scavenger receptor cysteine-rich domains is encoded by a separate exon. Fluorescence in situ hybridization studies and screening of a chromosome-specific YAC (yeast artificial chromosome) library revealed that the gene encoding CD6 is located on chromosome 11 at 11q13 in close proximity to the gene encoding the related molecule CD5 and within 600 kb of CD20. Analysis of mRNA transcripts encoding CD6 isolated from mitogen-activated PBMC and from B cells obtained from patients with chronic lymphocytic leukemia revealed the presence of at least five different CD6 transcripts. These transcripts arise via variable splicing of exons encoding the cytoplasmic domain of CD6. The existence of these isoforms suggests that signaling through CD6 could be regulated via alternative splicing of cytoplasmic encoding exons.

Asunto(s)

Antígenos CD/genética , Antígenos de Diferenciación de Linfocitos T/genética , Cromosomas Humanos Par 11 , Empalme Alternativo , Linfocitos B/fisiología , Secuencia de Bases , Cromosomas Artificiales de Levadura , Genes , Humanos , Intrones , Activación de Linfocitos , Datos de Secuencia Molecular , ARN Mensajero/genética , Mapeo Restrictivo

RESUMEN

CD5 is a member of a superfamily of proteins which contain one or more extracellular domains homologous to the type I macrophage Scavenger Receptor cysteine-rich (SRCR) domain. The extracellular region of CD5 is composed of three SRCR domains (D1, D2, D3). Murine CD5 (mCD5) is polymorphic (Ly-1.1 and Ly-1.2 alleles), however, the only murine CD5 gene characterized to date encodes the Ly-1.2 allele (mCD5.2). Likewise, the domain specificity of many of the available anti-mCD5 mAb recognizing either Ly-1.1 or Ly-1.2 or both has not been examined. Herein we describe the isolation and characterization of cDNA encoding the Ly1.1 allele (mCD5.1) and map the location and molecular nature of the mCD5 allelic variation. We also determined which SRCR domain of mCD5 is recognized by a panel of anti-mCD5 mAb. The mCD5.1 protein differs from mCD5.2 in only three amino acids, all of which map to the most amino terminal SRCR domain (D1) of mCD5. An additional seven silent substitutions were observed in the nucleotide sequence encoding mCD5 D1, D2 and transmembrane domains. Immunoglobulin (Ig) fusion proteins consisting of various combinations of mCD5.1 or mCD5.2 SRCR domains were produced and used to determine that allele specific mAb bound to D1, confirming sequence data. MAb against monomorphic determinants on mCD5 bound to each mCD5D11g.

Asunto(s)

Antígenos CD5/genética , Epítopos/genética , Animales , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Antígenos CD5/inmunología , Clonación Molecular , Cisteína , ADN Complementario , Epítopos/inmunología , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Ratas , Receptores Inmunológicos/genética , Receptores Depuradores

RESUMEN

Human CD6 is a cell surface protein expressed by thymocytes, mature T cells, a subset of B cells and certain cells of the brain. On human T cells, CD6 has been shown to act as a co-stimulatory molecule which modulates T cell receptor (TCR)-mediated T cell activation. To study further the recently identified mouse CD6 (mCD6), we generated and characterized a set of anti-mCD6 mAb. Anti-mCD6 mAb recognizing the mCD6 scavenger receptor cysteine-rich (SRCR) extracellular domains 1 and 3 were identified. mAb against SRCR domain 3, but not domain 1, inhibited the interaction of CD6 with a recently identified ligand, activated leukocyte cell adhesion molecule (ALCAM). Immunohistochemical analysis indicated that mCD6 expression was largely localized to the T cell areas of lymphoid tissue and, as previously reported in the human, CD6 was also expressed by neurons. CD6 was highly expressed on mouse T cells isolated from the spleen, lymph node and thymus as demonstrated by two-color immunofluorescence analysis. The CD4+ and CD8+ cells in these lymphoid compartments expressed similar levels of CD6. Immunoprecipitation studies showed that mouse thymocytes predominantly express a CD6 isoform of approximately 130 kDa, while splenocytes predominantly express a CD6 isoform of approximately 100 kDa. Anti-mCD6 mAb enhanced allogeneic mixed leukocyte reactions (MLR), indicating that CD6-ALCAM interactions may regulate the proliferative capacity of T cells.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Molécula de Adhesión Celular del Leucocito Activado , Animales , Anticuerpos Monoclonales/farmacología , Reacciones Antígeno-Anticuerpo/efectos de los fármacos , Glicoproteínas/metabolismo , Humanos , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Especificidad de la Especie

RESUMEN

Binding studies with a CD6 immunoglobulin fusion protein (CD6 Rg) resulted in the identification and cloning of a CD6 ligand. This ligand was found to be a member of the immunoglobulin supergene family and was named ALCAM (activated leukocyte cell adhesion molecule). Cell adhesion assays showed that CD6-ALCAM interactions mediate thymocyte-thymic epithelium cell binding. ALCAM is also expressed by activated leukocytes and neurons and may be involved in interactions between T cells and activated leukocytes and between cells of the immune and nervous systems, respectively. Herein we describe the preparation of domain-specific murine CD6 Rg fusion proteins and show that the membrane-proximal SRCR (scavenger receptor cysteine-rich) domain of CD6 contains the ALCAM binding site. We also show that mAbs which bind to this domain preferentially block CD6-ALCAM binding. These results demonstrate that the membrane-proximal SRCR domain of CD6 is necessary for CD6 binding to ALCAM and provide the first direct evidence for the interaction of an SRCR domain with a ligand.

Asunto(s)

Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Moléculas de Adhesión Celular/metabolismo , Glicoproteínas/metabolismo , Proteínas de la Membrana , Receptores Inmunológicos/metabolismo , Receptores de Lipoproteína , Molécula de Adhesión Celular del Leucocito Activado , Anticuerpos Monoclonales/farmacología , Especificidad de Anticuerpos , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Diferenciación de Linfocitos T/inmunología , Sitios de Unión , Epítopos , Humanos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica/efectos de los fármacos , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptores Depuradores , Proteínas Recombinantes de Fusión/metabolismo , Receptores Depuradores de Clase B , Relación Estructura-Actividad

RESUMEN

Clustering of beta 1-integrins on adherent cells with antibodies or ligands results in increased tyrosine phosphorylation and activation of a novel focal adhesion tyrosine kinase, pp125FAK. The genes encoding pp125FAK have been cloned previously from both chicken and mouse cDNA libraries, and the deduced amino acid sequences are nearly identical (94%). Two synthetic peptides derived from sequences at the carboxyl terminus of chicken pp125FAK were conjugated to ovalbumin to generate rabbit heteroantisera. Human pp125FAK was immunodetected in both T and B lymphocytes with these antisera. A basal state of pp125FAK tyrosine phosphorylation was observed in T and B lymphocytes, and its expression level was in general augmented among human T- and B-cell leukemia/lymphoma lines. Additionally, the full-length sequence of human T-cell pp125FAK (huT-FAK) was derived from a Jurkat T-cell cDNA library. huT-FAK is structurally identical with both mouse and chicken FAK, and shares 95% amino acid identity with chicken pp125FAK and has 97% homology with the mouse sequence. This high degree of evolutionary conservation between species suggests that pp125FAK is likely to have a crucial function in the cell. Expression of the full-length huT-FAK gene in COS cells showed an immunologically indistinct human pp125FAK protein compared with the endogenous primate pp125FAK. Taken together, the data indicate that this structurally conserved human T-cell pp125FAK likely functions in T- and B-cell lineages, and its altered expression in human lymphocyte tumor cell lines may contribute to their transformed phenotype.

Asunto(s)

Linfocitos B/enzimología , Moléculas de Adhesión Celular/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Linfocitos T/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Clonación Molecular , ADN Complementario/genética , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Expresión Génica , Humanos , Técnicas In Vitro , Leucemia de Células B/enzimología , Leucemia de Células T/enzimología , Ratones , Datos de Secuencia Molecular , Fosfotirosina , ARN Mensajero/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Células Tumorales Cultivadas , Tirosina/análogos & derivados , Tirosina/metabolismo

RESUMEN

This report describes the isolation and recombinant expression of a cDNA clone encoding HER4, the fourth member of the human epidermal growth factor receptor (EGFR) family. The HER4/erbB4 gene encodes a 180-kDa transmembrane tyrosine kinase (HER4/p180erbB4) whose extracellular domain is most similar to the orphan receptor HER3/p160erbB3, whereas its cytoplasmic kinase domain exhibits 79% and 77% identity with EGFR and HER2/p185erbB2, respectively. HER4 is most predominantly expressed in several breast carcinoma cell lines, and in normal skeletal muscle, heart, pituitary, brain, and cerebellum. In addition, we describe the partial purification of a heparin-binding HER4-stimulatory factor from HepG2 cells. This protein was found to specifically stimulate the intrinsic tyrosine kinase activity of HER4/p180erbB4 while having no direct effect on the phosphorylation of EGFR, HER2, or HER3. Furthermore, this heparin-binding protein induces phenotypic differentiation, and tyrosine phosphorylation, of a human mammary tumor cell line that overexpresses both HER4 and HER2. These findings suggest that this ligand-receptor interaction may play a role in the growth and differentiation of some normal and transformed cells.

Asunto(s)

Receptores ErbB/genética , Receptores ErbB/fisiología , Proteínas Tirosina Quinasas/genética , Receptores de Superficie Celular/genética , Secuencia de Bases , Células , Clonación Molecular , Receptores ErbB/metabolismo , Expresión Génica , Ligandos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Proteínas Tirosina Quinasas/metabolismo , ARN Mensajero/genética , Receptor ErbB-4 , Receptores de Superficie Celular/fisiología , Alineación de Secuencia , Transducción de Señal , Distribución Tisular

RESUMEN

Epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and amphiregulin are structurally and functionally related growth regulatory proteins. These secreted polypeptides all bind to the 170-kDa cell-surface EGF receptor, activating its intrinsic kinase activity. However, amphiregulin exhibits different activities than EGF and TGF-alpha in a number of biological assays. Amphiregulin only partially competes with EGF for binding EGF receptor, and amphiregulin does not induce anchorage-independent growth of normal rat kidney cells (NRK) in the presence of TGF-beta. Amphiregulin also appears to abrogate the stimulatory effect of TGF-alpha on the growth of several aggressive epithelial carcinomas that overexpress EGF receptor. These findings suggest that amphiregulin may interact with a separate receptor in certain cell types. Here we report the cloning of another member of the human EGF receptor (HER) family of receptor tyrosine kinases, which we have named "HER3/ERRB3." The cDNA was isolated from a human carcinoma cell line, and its 6-kilobase transcript was identified in various human tissues. We have generated peptide-specific antisera that recognizes the 160-kDa HER3 protein when transiently expressed in COS cells. These reagents will allow us to determine whether HER3 binds amphiregulin or other growth regulatory proteins and what role HER3 protein plays in the regulation of cell growth.

Asunto(s)

Receptores ErbB/genética , Expresión Génica , Genes , Proteínas Tirosina Quinasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular/métodos , Humanos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Conformación Proteica , Homología de Secuencia de Ácido Nucleico , Transfección , Células Tumorales Cultivadas/enzimología