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M Streuli

Publications and source records attributed to M Streuli.

At least 37 records · Page 2Linked to original sources
PubMed1994

Specific interaction of the CD45 protein-tyrosine phosphatase with tyrosine-phosphorylated CD3 zeta chain.

The CD45 transmembrane protein-tyrosine phosphatase (PTPase, EC 3.1.3.48) plays an essential role in T-cell activation by activating the Lck and/or Fyn protein-tyrosine kinases. However, numerous experiments have indicated that CD45 may have both stimulatory and inhibitory roles in T-cell activation. Thus, it is unlikely that the two kinases are the sole substrates of the CD45 PTPase. Furthermore, the complex regulation of the alternative splicing of the extracellular domain in various leukocyte lineages also suggests additional roles for the CD45 PTPase. To identify such functions, it is necessary to identify physiologically relevant substrates of the CD45 PTPase other than the two protein-tyrosine kinases. To this end, we searched for high-affinity substrates of the CD45 PTPase among the tyrosine-phosphorylated T-cell proteins by using purified glutathione S-transferase-CD45 fusion molecules. The enzymatically inactive CD45 C828S mutant protein, in which the cysteine residue at the catalytic center was changed to a serine residue, bound tightly to the phosphorylated CD3 zeta chain. This binding was specific to CD45 PTPase, as neither the leukocyte common antigen-related molecule (LAR) PTPase nor the CD45-LAR hybrid PTPases bound the phosphorylated CD3 zeta chain. Furthermore, phosphorylated CD3 zeta chain was preferentially dephosphorylated by the wild-type CD45 PTPase under conditions that did not significantly dephosphorylate other cellular proteins. Thus, the phosphorylated CD3 zeta chain is a specific and high-affinity substrate of the CD45 PTPase. These results suggest that CD45 is involved in the termination of the T-cell response via dephosphorylation of CD3 zeta chain.

CD3 Complex↗
PubMed1994

Genomic organization of the human LAR protein tyrosine phosphatase gene and alternative splicing in the extracellular fibronectin type-III domains.

The structure of the human leukocyte-common antigen-related molecule (LAR) protein tyrosine phosphatase gene was elucidated using phage and cosmid genomic DNA clones. The LAR gene is composed of 33 exons spanning over 85 kilobase pairs. Exon 2 encodes the signal sequence and the first four amino acids in the mature LAR protein. The three immunoglobulin-like domains are encoded by exons 3-7, and the eight fibronectin type III (Fn-III) domains by exons 8-17. Exons 18-22 encode the juxta-membrane and transmembrane domains, and exons 23-33 encode the two conserved tyrosine phosphatase domains and the entire 3'-untranslated region. Exon 1, which presumably encodes the 5'-untranslated sequence, has not been identified. Reverse transcription-polymerase chain reaction analysis revealed the alternative splicing of a mini-exon (exon 13) in the Fn-III domain 5 of human LAR and other related genes (rat LAR, rat PTP sigma, and human PTP delta). RNase protection analysis showed that the human LAR mRNA in which exon 13 is spliced-out is the major mRNA species in all cell lines examined. Reverse transcription-polymerase chain reaction analysis revealed further alternative splicing of LAR mRNA involving the Fn-III domains 4, 5, 6, and 7 in various combinations. These findings will facilitate the understanding of the physiological functions of the LAR extracellular domain.

Alternative Splicing↗
PubMed1994

Mutational analysis of proprotein processing, subunit association, and shedding of the LAR transmembrane protein tyrosine phosphatase.

The LAR transmembrane protein tyrosine phosphatase (PTPase) is expressed on the cell surface as a complex of two noncovalently associated subunits derived from a proprotein. The 150-kDa E-subunit contains most of the extracellular region, including the immunoglobulin-like and fibronectin type-III-like domains, whereas the 85-kDa P-subunit contains a short ectodomain, the transmembrane peptide, and the two intracellular PTPase domains. The LAR extracellular region is released from the cell surface, suggesting that shedding may be a mechanism to regulate LAR PTPase function. Functional regions necessary for LAR proprotein processing, subunit association, and shedding were determined by analyzing the effect of amino acid substitutions of residues surrounding the cleavage site and scanning the P-subunit ectodomain. Three amino acid residues were identified, two within a penta-arginine sequence and one C-terminal to the cleavage site, that are essential for efficient LAR proprotein cleavage. Several noncontiguous amino acid residues were also identified that play an essential role in LAR subunit association. LAR shedding is shown to be a consequence of proteolytic cleavage at a second site within the P-subunit ectodomain near the transmembrane peptide.

Amino Acid Sequence↗
PubMed1994

Interleukin 2 receptor gamma chain expression on resting and activated lymphoid cells.

The interleukin 2 receptor (IL-2R) is known to be comprised of at least three genetically distinct subunits termed alpha, beta, and gamma. These chains can be expressed individually or in various combinations resulting in distinct receptors with different affinities for IL-2. In contrast to alpha and beta, the cell surface expression of the gamma chain protein previously has not been well-characterized. To examine cell surface expression of IL-2R gamma on hematopoietic cells, we developed two new monoclonal antibodies (mAbs) specific for this protein. Both 1A11 (immunoglobulin [IgG1]) and 3G11 (IgM) specifically reacted with murine cells transfected with IL-2R gamma cDNA, and immunoprecipitation studies indicated that both antibodies precipitated a protein of approximately 62-65 kD. Scatchard analysis of IL-2 binding to murine cells transfected with cDNA-encoding combinations of IL-2R components demonstrated that neither beta nor gamma chain bind IL-2 with measurable affinity, but coexpression of both beta and gamma is sufficient to form an intermediate affinity receptor. In the absence of gamma chain, beta chain interacts with alpha chain to form a "pseudo-high" affinity receptor. In contrast, gamma chain does not appear capable of interacting with alpha in the absence of beta chain. Thus, gamma chain appears to interact only with beta, but beta chain is capable of interacting with both alpha and gamma. Using the newly developed mAbs to examine cell surface expression by immunofluorescence, resting T cells were found to express low levels of gamma chain without detectable alpha or beta. Early after mitogen stimulation, T cells expressed higher levels of alpha, beta, and gamma. However, at later time points, T cells expressed alpha and gamma in marked excess over beta. Thus, formation of high affinity IL-2R on activated T cells was primarily limited by beta chain expression. In contrast, resting natural killer (NK) cells constitutively expressed IL-2R beta without detectable alpha or gamma. After activation with either IL-2 or IL-12, expression of both alpha and gamma transiently increased and then returned to very low levels. Expression of functional IL-2R on resting and activated NK cells, therefore, appeared to be primarily limited by the expression of gamma chain. IL-2 binding studies with resting NK cells confirmed the results of immunofluorescence studies indicating the presence of very low numbers of intermediate affinity (beta gamma) receptors for IL-2 on these cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Intron-exon organization and chromosomal localization of the human TIA-1 gene.

TIA-1 is a 40-kDa cytotoxic granule-associated RNA-binding protein (p40-TIA-1), the expression of which is restricted to cytolytic lymphocytes. The major granule-associated species is a 15-kDa protein (p15-TIA-1) that seems to be derived from the carboxyl terminus of p40-TIA-1. Although some evidence suggests that p15-TIA-1 is derived from p40-TIA-1 by proteolytic processing, it is also possible that each isoform is derived from discrete mRNA initiated from alternative promoters within the TIA-1 gene. To learn more about the relationship between p15-TIA-1 and p40-TIA-1, we have determined the complete intron-exon organization of the TIA-1 gene. The gene consists of 13 exons separated by 12 intervening sequences and spans greater than 46 kb of DNA located on chromosome 2, band p13. The transcription start site of the mRNA transcript encoding p40-TIA-1 was identified by primer extension and S1 mapping analysis. The putative promoter region preceding this transcription start site stimulated the expression of a reporter gene in transfected Jurkat and YT cells. Attempts to identify a second promoter capable of initiating an mRNA encoding p15-TIA-1 were unsuccessful, supporting the possibility that p15-TIA-1 can be derived from p40-TIA-1 proteolytically. Granule-associated serine proteases that have been implicated in cytolytic lymphocyte killing (granzyme A and granzyme B) were unable to cleave TIA-1, suggesting that processing may occur before TIA-1 enters the cytotoxic granule.

Amino Acid Sequence↗
PubMed1994

Identification of amino acids at the junction of exons 3 and 7 that are used for the generation of glycosylation-related human CD45RO and CD45RO-like antigen specificities.

The CD45 transmembrane protein tyrosine phosphatase plays an essential role in lymphocyte activation. In humans, CD45 is composed of five isoforms that are generated by alternative splicing of three exons of a common precursor mRNA. Expression of the smallest molecular mass 180-kD CD45 isoform (CD45-O) results from splicing out of exons 4(A), 5(B), and 6(C), which encode peptide regions near the NH2 terminus, and is regulated during T cell maturation and activation. Two monoclonal antibodies (mAb), UCHL1 (anti-CD45RO) and A6 (anti-CD45RO-like), were studied that selectively bind to murine transfectant cells expressing the human CD45-O isoform. The anti-CD45RO-like A6 mAb, but not the anti-CD45RO UCHL1 mAb, also weakly reacted with transfectant cells expressing the human CD45 isoforms that contained exons 4 and 5(AB), or exon 5(B) encoded sequences. The structural basis of the antigen specificities of these two different human anti-CD45RO mAbs was investigated at the molecular level by using potential glycosylation-defective CD45-O isoform variants containing amino acid substitutions at the junction of exons 3 and 7. Replacement of the threonine residue at position 8 (last amino acid encoded in exon 3 and a putative O-linked carbohydrate anchorage site) by an alanine, completely abrogated the reactivity of the UCHL1 mAb, but did not affect that of the A6 mAb. Conversely, replacement of either the asparagine at position 174 or the serine at position 176 (the first two putative carbohydrate anchorage sites in exon 7) by alanine, abrogated the reactivity of the A6 mAb, but not that of the UCHL1 mAb. Both the UCHL1 and A6 epitopes were dependent on the presence of O-linked carbohydrates; and the UCHL1, but not the A6 epitope, was dependent on the presence of sialic acid. These results demonstrate a pivotal role for the amino acids encoded at the junction of exons 3 and 7 for the generation of glycosylation-related CD45RO epitopes that are expressed in a cell lineage- and activation-regulated fashion.

Alternative Splicing↗
PubMed1992

Identification and functional characterization of a TIA-1-related nucleolysin.

We recently reported the molecular cloning of a cytotoxic granule-associated RNA-binding protein designated TIA-1. The ability of recombinant TIA-1 to induce DNA fragmentation in permeabilized cells suggested that this protein is the granule component responsible for inducing apoptosis in cytolytic lymphocyte (CTL) targets. Here we report the characterization of a cDNA encoding a TIA-1-related protein designated TIAR. The deduced amino acid sequence of TIAR reveals it to be a 42-kDa protein possessing three RNA-binding domains and a carboxyl-terminal auxiliary domain. Although the RNA-binding domains of TIA-1 and TIAR share greater than 85% amino acid homology, their carboxyl-terminal auxiliary domains are only 51% homologous. The carboxyl terminus of TIAR contains a lysosome-targeting motif, indicating that TIAR is probably a cytotoxic granule-associated protein. Like TIA-1, purified recombinant TIAR induced DNA fragmentation in permeabilized target cells. Although immunoblotting analysis of post-nuclear supernatants revealed TIA-1 protein to be restricted to CTLs, PCR analysis revealed the expression of TIA-1 and TIAR mRNA transcripts in a wide variety of cell types. Our data suggest that the granules of CTLs contain at least two candidate nucleolysins involved in CTL killing.

Amino Acid Sequence↗
PubMed1992

Purification and characterization of the catalytic domains of the human receptor-linked protein tyrosine phosphatases HPTP beta, leukocyte common antigen (LCA), and leukocyte common antigen-related molecule (LAR).

Human HPTP beta, leukocyte common antigen (LCA), and leukocyte common antigen-related molecule (LAR) are transmembrane receptor-like proteins whose cytoplasmic regions contain either one (HPTP beta) or two (LCA and LAR) domains that are homologous to protein tyrosine phosphatases (PTPases). Whereas the membrane-proximal domain 1 has enzymatic activity, the membrane-distal domain 2 of both LCA and LAR has no detectable catalytic activity. The cytoplasmic regions of HPTP beta, LCA, and LAR were expressed in Escherichia coli and purified to greater than 90% purity. Modulatory effects of various low molecular weight compounds and homo- and copolymers of amino acids were examined. Several polypeptides that contain a high proportion of tyrosine were strongly inhibitory to these PTPases. To determine a possible role for the LAR domain 2, the properties of recombinant LAR PTPases containing both domains 1 and 2 (LAR-D1D2) or only domain 1 (LAR-D1) were compared. In nearly all aspects examined, LAR-D1 and LAR-D1D2 were indistinguishable. However, polycationic polypeptides strongly stimulated the PTPase activity of LAR-D1D2, but not LAR-D1, using the peptide substrate Raytide. Thus, basic polypeptides seem to indirectly alter the catalytic activity of domain 1 by interacting with domain 2. This result suggests that domain 2 has a regulatory function.

Amino Acid Sequence↗
PubMed1992

The alternative splicing of the CD45 tyrosine phosphatase is controlled by negative regulatory trans-acting splicing factors.

CD45, a receptor-type protein tyrosine phosphatase involved in lymphocyte activation, consists of five isoforms generated by alternative mRNA splicing. The pattern of CD45 splicing depends upon cell type, state of activation, and thymic development. We previously showed that mouse B and thymocyte cell lines transfected with a human CD45 minigene construct appropriately splice the alternative exons, demonstrating the presence of trans-acting tissue-specific splicing factors. To study the regulation of CD45 alternative splicing, cells having different splicing patterns were transiently fused, and mRNA was analyzed using reverse transcription-polymerase chain reaction. Human B cells, normally producing only the largest CD45 mRNAs, could splice out the alternative exons after fusion with mouse thymocytes. In contrast, the splicing pattern of human T cell lines (smaller CD45 isoforms) was unaltered by fusion with mouse B cells. This suggests that cells expressing the smallest CD45 isoform contain negatively acting trans-factor(s) that allow the alternative exons to be skipped, and that the full length isoform containing all three alternative exons is the default pattern of splicing. In agreement, incubation of thymocytes or T cell subsets with cycloheximide increased mRNA corresponding to the larger isoforms. CD45 isoform expression can now be interpreted in terms of the presence or absence of negative regulatory trans-acting splicing factors.

Animals↗
PubMed1992

Expression of the receptor-linked protein tyrosine phosphatase LAR: proteolytic cleavage and shedding of the CAM-like extracellular region.

The human transmembrane molecule LAR is a protein tyrosine phosphatase (PTPase) with a cell adhesion molecule-like extracellular receptor region. The structure of LAR hinted at its involvement in the regulation of tyrosine phosphorylation through cell-cell or cell-matrix interactions. We show here that LAR is expressed on the cell surface as a complex of two non-covalently associated subunits derived from a proprotein. The LAR E-subunit contains the cell adhesion molecule-like receptor region, while the LAR P-subunit contains a short segment of the extracellular region, the transmembrane peptide and the cytoplasmic PTPase domains. Proprotein processing occurs intracellularly. Analysis of LAR mutants suggested that cleavage occurs in the LAR extracellular region at a paired basic amino acid site by a subtilisin-like endoprotease. A single amino acid substitution at this site blocked LAR proprotein cleavage. The LAR E-subunit is shed during cell growth, suggesting that LAR receptor shedding may be a mechanism for regulating PTPase function. The use of immunohistochemistry techniques on human tissues demonstrated the expression of LAR by various cell lineages, including epithelial cells, smooth muscle cells and cardiac myocytes. The LAR gene is mapped to chromosome 1, region p32-33, which contains candidate tumor suppressor genes.

Amino Acid Sequence↗
PubMed1992

Activation of T cells through a T cell-specific epitope of CD45.

The 180- and 190-kDa isoforms of CD45 are preferentially expressed on the helper inducer (memory) subset of CD4 cells. In order to generate monoclonal antibodies against the extracellular domains of these isoforms and determine whether they could regulate the function and activation of these cells, we developed a mAb, anti-4H2D, by immunizing Balb/c mice with an isogenic mouse pre-B cell line expressing the human 190-kDa CD45 isoform. Anti-4H2D reacts with approximately 60% of T cells, 70% of CD4 cells, and 60% of CD8 cells. The CD4 cell population defined by this mAb corresponds functionally and phenotypically to that defined by the CD45RO+CD29+ subset. Western blotting demonstrated that anti-4H2D reacts primarily with the 190-kDa isoform of CD45 and to a minor extent, the 205- and 180-kDa CD45 isoforms. Interestingly, this mAb reacted with only a subpopulation of mature thymocytes and peripheral T cells, despite the fact that the 190-kDa CD45 isoform, as well as CD45RO and CD29, is more widely distributed on cells of hematopoietic origin. The 4H2D epitope was neuraminidase sensitive, indicating that anti-4H2D reacts with a carbohydrate epitope which is present on only a subset of the T cells containing the 190-kDa CD45 isoform epitopes. Functional studies showed that soluble anti-4H2D augmented T cell proliferation induced by the CD2 and CD3 pathways, and treatment of T cells with this mAb up-regulated [Ca2+]i flux induced by both anti-CD2 and anti-CD3 mAbs. These results suggest that the 190-kDa CD45 isoform on human CD4 cells is heterogeneous and that the 190-kDa isoform recognized by anti-4H2D regulates the function and activation of CD4 helper T cells.

Antibodies, Monoclonal↗
PubMed1991

A polyadenylate binding protein localized to the granules of cytolytic lymphocytes induces DNA fragmentation in target cells.

Cytolytic lymphocytes (CTLs) are characterized by their inclusion of cytoplasmic granules containing effector molecules such as perforin and the serine proteases. Here we describe the cDNA cloning and functional characterization of two related isoforms of a cytolytic granule protein designated TIA-1. Sequence analysis reveals that the 40 kd TIA-1 isoform (rp40-TIA-1) is structurally related to the poly(A)-binding proteins, possessing three RNA-binding domains and a carboxy-terminal, glutamine-rich auxiliary domain. The 15 kd TIA-1 isoform, the major species present in cytolytic granules, appears to be derived from the carboxy-terminal auxiliary domain of rp40-TIA-1 by proteolytic processing. Both natural and recombinant TIA-1 were found to induce DNA fragmentation in digitonin permeabilized thymocytes, suggesting that these molecules may be the granule components responsible for inducing apoptosis in CTL targets.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of temperature-sensitive and thermostable mutants of the human receptor-like protein tyrosine phosphatase LAR.

Human LAR is a transmembrane receptor-like protein whose cytoplasmic region contains two tandemly duplicated domains homologous to protein tyrosine phosphatases (PTPases). Whereas the membrane-proximal domain I has enzymatic activity, the membrane-distal domain II has no apparent catalytic activity but seems to have a regulatory function. In order to study structure-function relationships of the LAR PTPase, LAR domain I was expressed in Escherichia coli, and mutants that have reduced catalytic activity or reduced thermostability were isolated and characterized. We isolated 18 unique hydroxylamine-induced missense mutations in the LAR domain I segment, of which three were temperature-sensitive. Five additional temperature-sensitive mutations were isolated using N-methyl-N'-nitro-N-nitrosoguanidine. All eight temperature-sensitive mutations are confined within a short segment of the LAR domain I sequence between amino acid positions 1329 and 1407. To examine whether this region is particularly prone to temperature-sensitive mutations, tyrosine at amino acid position 1379 was changed to a phenylalanine by oligonucleotide-directed mutagenesis. This mutant, Y1379-F, was indeed temperature-sensitive. We also isolated a revertant of a temperature-sensitive mutant. The revertant contained a second-site mutation (C1446-Y) that suppresses several temperature-sensitive mutations and also enhances the folding of LAR protein produced in E. coli.

Amino Acid Sequence↗
PubMed1990

Effect of activation of protein kinase C on CD45 isoform expression and CD45 protein tyrosine phosphatase activity in T cells.

The T200/leukocyte common antigen (CD45) is a family of at least five large-molecular weight glycoproteins, which are differentially expressed on T cell subsets. The CD45 antigen consists of a variable heavily glycosylated exterior domain, a single membrane-spanning region, and a large cytoplasmic domain that has protein tyrosine phosphatase (PTPase) activity. In this study, we examined the effects of activation of protein kinase C (PKC) on the phosphorylation and expression of CD45 isoforms and PTPase activity in human T cells. After activation of PKC by phorbol 12-myristate 13-acetate (PMA), CD45RA expression rapidly increased within the first 24 h, whereas CD45R0 expression did not change within this time. However by 48 h, expression of CD45R0 also began to increase. Metabolic labeling showed that the rapid increment in CD45RA expression observed after PMA stimulation is primarily due to increased de novo synthesis of the 205-kDa and not the 220-kDa molecule. PMA treatment resulted in the phosphorylation of each CD45 isoform to a degree corresponding to its relative surface expression. Significantly, we found that the phosphorylation of CD45 by PKC activation down-regulated CD45 PTPase activity.

Antigens, Differentiation↗
PubMed1990

Distinct functional roles of the two intracellular phosphatase like domains of the receptor-linked protein tyrosine phosphatases LCA and LAR.

Protein tyrosine phosphorylation is regulated by both protein tyrosine kinases and protein tyrosine phosphatases (PTPases). Recently, the structures of a family of PTPases have been described. In order to study the structure-function relationships of receptor-linked PTPases, we analyzed the effects of deletion and point mutations within the cytoplasmic region of the receptor-linked PTPases, LCA and LAR. We show that the first of the two domains has enzyme activity by itself, and that one cysteine residue in the first domain of both LCA and LAR is absolutely required for activity. The second PTPase like domains do not have detectable catalytic activity using a variety of substrates, but sequences within the second domains influence substrate specificity. The functional significance of a stretch of 10 highly conserved amino acid residues surrounding the critical cysteine residue located in the first domain of LAR was assessed. At most positions, any substitution severely reduced enzyme activity, while missense mutations at the other positions tested could be tolerated to varying degrees depending on the amino acid substitution. It is suggested that this stretch of amino acids may be part of the catalytic center of PTPases.

Amino Acid Sequence↗