Jak3 activation in human lymphocyte precursor cells.

Although expression of the Jak3 tyrosine kinase in T lymphocytes has been thought to be restricted to mature, activated cells, mutations of Jak3 can lead to the development of a human severe combined immunodeficiency (SCID) characterized by an absence of peripheral T lymphocytes. We therefore examined in detail the expression of Jak3 throughout human T cell differentiation and show that Jak3 is in fact present throughout the entire developmental process, with high levels expressed in thymocytes. Jak3 is highly expressed in double negative (CD4- CD8-) cells, one of the earliest stages of thymocyte differentiation, and can be activated via the IL-7 receptor. IL-7 is known to stimulate thymocyte proliferation and initiate re-arrangement of the T cell receptor (TCR) beta gene, suggesting that the failure of mutated Jak3 proteins to transduce this signal may be responsible for failures in T cell development. While Jak3 SCID patients possess mature peripheral B cells, we demonstrate that the Jak3 tyrosine kinase is also expressed in human pre-B cells and can be activated by the pre-B cell growth factor IL-7.

Human immune disorder arising from mutation of the alpha chain of the interleukin-2 receptor.

Profound cellular immunodeficiency occurs as the result of mutations in proteins involved in both the differentiation and function of mature lymphoid cells. We describe here a novel human immune aberration arising from a truncation mutation of the interleukin-2 receptor alpha chain (CD25), a subunit of the tripartite high-affinity receptor for interleukin 2. This immunodeficiency is characterized by decreased numbers of peripheral T cells displaying abnormal proliferation but normal B cell development. Extensive lymphocytic infiltration of tissues, including lung, liver, gut, and bone, is observed, accompanied by tissue atrophy and inflammation. Although mature T cells are present, the absence of CD25 does affect the differentiation of thymocytes. While displaying normal development of CD2, CD3, CD4, and CD8 expression, CD25-deficient cortical thymocytes do not express CD1, and furthermore they fail to normally down-regulate levels of the anti-apoptotic protein bcl-2.

JAK3 protein tyrosine kinase mediates interleukin-7-induced activation of phosphatidylinositol-3' kinase.

The interleukin-7 (IL-7) receptor is expressed throughout T-cell differentiation and, although lacking a tyrosine kinase domain, mediates tyrosine phosphorylation in T cells. We have identified IL-7-induced activation of three cyoplasmic tyrosine kinases in T cells, Jak1, Jak3, and the src-like kinase p56lck. Many members of the cytokine receptor superfamily activate the Jak protein tyrosine kinase family, with resultant phosphorylation of the Stat transcriptional activator factors. We describe here a novel function of the Jak kinases, because Jak kinase activity is not only required for Stat activation but also for P13 kinase response to IL-7 in human T cells. We show that IL-7 receptor-mediated Jak activation can occur independently of p56lck activity. IL-7-induced P13 kinase activation, mediated by tyrosine phosphorylation of the P13 kinase p85 subunit, is essential to the IL-7 proliferative signal and also occurs in the absence of src family kinase activity. Jak3 is found associated with the p85 subunit of P13 kinase in an IL-7-responsive manner in T cells and appears to regulate IL-7-induced P13 kinase activation by mediating tyrosine phosphorylation of the p85 subunit. Specific inhibition of IL-7-induced Jak kinase activity ablates p85 tyrosine phosphorylation, subsequent P13 kinase activation, and, ultimately, proliferation. The ability to regulate P13 kinase activity indicates a more generalized role for the Jak family than activation of gene transcription via the Stat family in cytokine receptor signal transduction.

Activation of phosphatidylinositol-3 kinase by ligation of the interleukin-7 receptor on human thymocytes.

Interleukin-7 (IL-7) is a glycoprotein that regulates lymphocyte precursor growth and differentiation. However, the exact mechanism whereby the IL-7 receptor (IL-7R) mediates these cell growth signals remains unknown. One of the earliest metabolic events linked to mitogenic responses in other growth factor receptor systems is the activation of phosphatidylinositol-3 kinase (PI-3 kinase). We demonstrate here that ligation of the IL-7R results in dose- and time-dependent increases in PI-3 kinase activity. These results suggest that PI-3 kinase is involved in signal transduction via the IL-7R in human thymocytes.

Interleukin 7 receptor mediates the activation of phosphatidylinositol-3 kinase in human B-cell precursors.

Interleukin 7 appears to be central to promoting growth and differentiation of lymphocyte precursors. Pro-B cells, as well as pre-pre-B cells, but not mature cells express the IL-7 receptor suggesting an important role for IL-7 during these early stages of B cell maturation. Indeed, IL-7 was shown to promote not only growth but also immunoglobulin gene rearrangement in these cells. However, the mechanism by which IL-7 receptor transduces these growth signals remains elusive. In an attempt to characterize a pathway of signal transduction via the IL-7 receptor, we have demonstrated here that ligation of the IL-7 receptor is associated with increased activity of PI-3 kinase. PI-3 kinase products were detected by TLC and their identity confirmed by HPLC. Further, by in vivo labeling intact cells we have shown that IL-7 receptor mediates the activation of PI-3 kinase.

CD44 in human placenta: localization and binding to hyaluronic acid.

CD44, a receptor for hyaluronic acid (HA), has been identified in the stroma of stem and terminal chorionic villi of human term placenta. The CD44 glycoprotein antigen, isolated from placenta by affinity to monoclonal antibody (mAb) 50B4, consisted mainly of species of M(r) 85,000 and 200,000. Radiolabelled CD44 bound specifically to HA attached to plastic, predominantly via the M(r) 85,000 species; this binding was inhibited by soluble HA and hyaluronidase. The binding of CD44 to HA was also inhibited by mAb 50B4 and IM7.8.1, which recognize epitopes of cluster I and II respectively, but was not blocked by a polyclonal antibody to peptide 18-30 of the B loop (residues 12-101). These results suggest that the portion of the B loop of CD44 implicated in the binding to HA is between amino acids 31-101 and that epitopes located outside the B loop, such as that recognized by mAb IM7.8.1 (between residues 132-215), contribute to this interaction. The presence of a functional CD44 molecule in the human term placenta suggest a role for this molecule in situ in the stabilization and orientation of HA network important in the maintenance of the structural integrity of the placenta.

CD10 and CD44 genes of leukemic cells and malignant cell lines show no evidence of transformation-related alterations.

The expression of CD10/CALLA is associated primarily with childhood leukemia of pre-B lymphocyte phenotype. We have compared the hybridization pattern of the CALLA gene from leukemic and normal cells digested with several restriction enzymes. No alterations were noticed with Eco RI, Sac I, Pvu II, Eco RV, Hind III, and Msp I. Since CALLA is also found on other malignancies, we analyzed DNA samples prepared from cell lines derived from leukemia, lymphoma, glioblastoma, retinoblastoma, and neuroblastoma. Normal restriction patterns were observed for all the lines regardless of their CALLA phenotype. Having demonstrated previously that CALLA was structurally identical to neutral endopeptidase 3.4.24.11 (NEP), we have now established a correlation between surface expression of CALLA and NEP activity on leukemia samples and on several cell lines. Malignant cells tested expressed a functionally active enzyme and no gross alteration was present in the CALLA gene. The CD44 gene is expressed on most cells of hemopoietic origin and on greater than 95% of cases of acute lymphoblastic leukemia and acute myeloblastic leukemia studied. It is also expressed on normal astrocytes and on malignant cells of glioma/astrocytoma types. We now report that a similar pattern of hybridization was observed with Sac I, Pvu II, and Eco RI for leukemic samples, normal cells, and malignant cell lines. A polymorphism was recently detected for CD44 using Hind III; leukemic cells and malignant lines also showed this normal polymorphism. Thus no deletion or insertion could be detected in the CD44 gene of leukemic cells and malignant lines, suggesting that no gross DNA alterations were involved. The correlation between surface expression and enzymatic activity of CD10/CALLA and the expression of CD44 on a variety of malignant cells would suggest that the structure and function of these two gene products are probably not altered by the process of transformation.

Structural microheterogeneity of the muscarinic cholinergic receptor is not related to functional diversity identified by differences in affinity for pirenzepine.

The muscarinic receptor for acetylcholine shows a diversity in ligand binding properties and effector mechanisms which have suggested the existence of two subtypes (M1 and M2), to which the selective antagonist pirenzepine binds with markedly different affinities. The receptor from rat brain, covalently labelled with the alkylating antagonist tritiated propylbenzilylcholine mustard, displays a structural microheterogeneity on electrophoresis, covering the region of apparent molecular weight 66,000-76,000, with dominant components at 68,000 and 73,000. Selective inhibition by pirenzepine of labelling of the M1 receptor with tritiated mustard has been analysed on fluorographs of sodium dodecyl sulphate-polyacrylamide gels and shown to cause a uniform reduction in radioactive labelling of the broad receptor peak, rather than selectively inhibiting either the high- or low-molecular-weight regions of the band. It is further shown that although this receptor microheterogeneity is found for each of four brain regions studied, it is not found for the heart receptor, which gives a discrete labelled band of apparent molecular weight 72,000. It is therefore suggested that the structural microheterogeneity is the result of tissue-specific, posttranslational modification of the molecule, such as glycosylation, and is not directly related to the functional diversity of the receptor.

Muscarinic cholinergic receptor of rat brain. Factors influencing migration in electrophoresis and gel filtration in sodium dodecyl sulphate.

The muscarinic cholinergic receptor present in synaptosomal membranes of rat brain was covalently labelled with the alkylating muscarinic antagonist, tritiated propylbenzilylcholine mustard. The labelled receptor was then solubilized in sodium deoxycholate and sodium dodecyl sulphate, and its migration in polyacrylamide gel electrophoresis and gel filtration in the presence of sodium dodecyl sulphate analysed. Provided both proteolysis and inter-chain disulphide bond formation were vigorously prevented, the receptor from rat forebrain (cerebral cortex plus caudate putamen) migrated, in sodium dodecyl sulphate/polyacrylamide gel electrophoresis, as a broad band of apparent Mr 66000-76000. Two dominantly labelled polypeptides, of apparent Mr 68000 and 73000, could be distinguished as the major components of this band. These multiple species seen in electrophoresis may reflect a structural diversity related to the different binding properties, and modes of action, of this receptor. In electrophoresis using discontinuous buffer systems the labelled receptor readily formed intermolecular disulphide bonds and so aggregated. In particular, if solubilized membranes were reduced with 2-mercaptoethanol, and reformation of disulphide bonds during electrophoresis not prevented, then formation of a dimeric species (apparent Mr 119000-128000) occurred. This probably explains previous reports in the literature of larger-Mr species seen in electrophoresis. During gel filtration, the receptor formed intra-chain disulphide bonds which produced conformational heterogeneity, leading to polydisperse migration. In addition, extensive proteolytic degradation of the receptor occurred due to a protease migrating slightly ahead of the receptor. Both effects were eliminated by alkylation of the solubilized membranes with iodoacetamide before gel filtration. Alkylated receptor migrated on Sephacryl S-300 in 0.5% sodium dodecyl sulphate with an equivalent Stokes' radius of 6.1 nm. This is identical to that of reduced ovalbumin, a molecule with an apparent Mr in gel electrophoresis of 43000. On a different gel matrix, TSK HW 55(S), the receptor migrated with a somewhat larger Stokes' radius, eluting just behind reduced bovine serum albumin (Stokes' radius 8.5 nm; apparent Mr in electrophoresis 67000). Thus the receptor appears to adsorb to the Sephacryl matrix, although even on the TSK gel the receptor eluted as a somewhat smaller protein than expected from its behaviour in gel electrophoresis. Solubilized, alkylated receptor, partly purified by gel filtration so that it was not degraded by endogenous proteases, was not cleaved by mild hydroxylamine treatment.(ABSTRACT TRUNCATED AT 400 WORDS)