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I S Trowbridge

Publications and source records attributed to I S Trowbridge.

At least 55 records · Page 3Linked to original sources
PubMed1990

Coclustering CD45 with CD4 or CD8 alters the phosphorylation and kinase activity of p56lck.

Antibody-mediated CD4 crosslinking results in increased tyrosine phosphorylation and tyrosine kinase activity of the associated p56lck. Treatment with anti-CD4 and anti-Ig also induced the phosphorylation of p56lck in a CD45- mutant cell line, indicating that the increase in phosphotyrosine content of p56lck is not the result of being sequestered from CD45 protein tyrosine phosphatase (PTPase). Antibody-mediated coclustering of CD45 with CD4 inhibited the anti-CD4-induced phosphorylation of p56lck on tyrosine and the concomitant increase in in vitro kinase activity. Similar results were obtained when CD45 was coclustered with CD8 on cytotoxic T cell lines. These observations provide strong evidence that p56lck is a substrate for CD45 in vivo and provide an assay to study the regulation and specificity of CD45 PTPase activity.

Antigen-Antibody Reactions↗
PubMed1990

Role of the human transferrin receptor cytoplasmic domain in endocytosis: localization of a specific signal sequence for internalization.

Wild-type and mutant human transferrin receptors have been expressed in chicken embryo fibroblasts using a helper-independent retroviral vector. The internalization of mutant human transferrin receptors, in which all but four of the 61 amino acids of the cytoplasmic domain had been deleted, was greatly impaired. However, when expressed at high levels, such "tailless" mutant receptors could provide chicken embryo fibroblasts with sufficient iron from diferric human transferrin to support a normal rate of growth. As the rate of recycling of the mutant receptors was not significantly different from wild-type receptors, an estimate of relative internalization rates could be obtained from the distribution of receptors inside the cell and on the cell surface under steady-state conditions. This analysis and the results of iron uptake studies both indicate that the efficiency of internalization of tailless mutant receptors is approximately 10% that of wild-type receptors. Further studies of a series of mutant receptors with different regions of the cytoplasmic domain deleted suggested that residues within a 10-amino acid region (amino acids 19-28) of the human transferrin receptor cytoplasmic domain are required for efficient endocytosis. Insertion of this region into the cytoplasmic domain of the tailless mutant receptors restored high efficiency endocytosis. The only tyrosine residue (Tyr 20) in the cytoplasmic domain of the human transferrin receptor is found within this 10-amino acid region. A mutant receptor containing glycine instead of tyrosine at position 20 was estimated to be approximately 20% as active as the wild-type receptor. We conclude that the cytoplasmic domain of the transferrin receptor contains a specific signal sequence located within amino acid residues 19-28 that determines high efficiency endocytosis. Further, Tyr 20 is an important element of that sequence.

Amino Acid Sequence↗
PubMed1990

p180, a novel recycling transmembrane glycoprotein with restricted cell type expression.

A 180-kilodalton (kDa) protein (p180) was identified among the antigens for a panel of monoclonal antibodies raised against human fibroblast cell surface proteins. Binding studies with 125I-Fab' fragments of an anti-p180 monoclonal antibody demonstrated that 10 to 30% of p180 was located on the plasma membrane and that the remaining 70 to 90% was on intracellular membranes. p180 was rapidly internalized from the cell surface at 37 degrees C, and kinetic analyses indicated that this was a constitutive process followed by the recycling of p180 back to the plasma membrane. Morphological studies demonstrated that on the cell surface p180 was concentrated in coated pits, whereas inside the cell it was found in endosomes as suggested by its colocalization with the transferrin receptor. Immunoblot analysis with a polyclonal antiserum raised against purified human protein showed that p180 has a restricted distribution with expression at high levels in fibroblast cultures and in tissues containing cells of mesodermal origin. A biochemical characterization of p180 showed it to be a transmembrane glycoprotein with an extracellular domain, which consists of approximately 30 kDa of complex oligosaccharides attached to at least 45 kDa of the protein core. The cytoplasmic domain of p180 was found to contain a serine residue(s) that was phosphorylated both in vivo and in vitro by activated protein kinase C. p180 was purified by subjecting solubilized membrane proteins from a human osteosarcoma cell line to immunoaffinity chromatography and gel filtration. The N-terminal sequence information obtained from the purified protein showed no homology to other known proteins. It was concluded that p180 may be a novel recycling receptor which is highly restricted in its expression to fibroblastlike cells.

Antibodies, Monoclonal↗
PubMed1989

Identification of the alternatively spliced exons of murine CD45 (T200) required for reactivity with B220 and other T200-restricted antibodies.

Cell lines expressing specific variants of murine CD45 (T200) were established by infection with retroviral constructs of four cDNAs encoding different forms of CD45. These lines were then used to determine which sequences encoded by alternatively spliced exons of CD45 were required to generate antigenic determinants recognized by anti-CD45 mAbs. The binding of two B220 antibodies (14.8 and RA32C2) to CD45 was dependent on the expression of the first alternatively spliced exon (exon A). A third B220 antibody, RA3-6B2, did not bind to any of the forms of CD45 expressed on fibroblasts. A newly defined anti-CD45R antibody, C363.16A, reacts with an antigenic site dependent upon the expression of the second alternatively spliced exon, exon B.

Animals↗
PubMed1989

Changes in CD45 isoform expression accompany antigen-induced murine T-cell activation.

Leukocytes express a family of plasma membrane proteins called CD45 or the leukocyte common antigen. Isoforms of various molecular masses, 180-240 kDa, are produced by alternative splicing and usage of three exons, named A, B, and C, that encode the N-terminal portion of the external domain. By using monoclonal antibodies that precipitate B exon-dependent and B exon-independent isoforms we find that both murine CD4+ and murine CD8+ T cells selectively down-regulate the B exon-dependent forms of CD45 during an immune response. This change was monitored by using fluorescence-activated cell sorter (FACS) analysis and immunoprecipitation from surface radioiodinated and metabolically labeled cells. The loss of the 190-kDa B exon-dependent isoform during T-cell activation is accompanied by an increased production of a 180-kDa form, which does not contain the B exon-encoded sequence. This accounts for our observation that the overall expression of CD45, as assessed by FACS analysis, does not change.

Animals↗
PubMed1989

Expression of CD45 alters phosphorylation of the lck-encoded tyrosine protein kinase in murine lymphoma T-cell lines.

CD45 is a family of high molecular weight leukocyte cell surface glycoproteins. Recently, two related subregions of the cytoplasmic domain of CD45 have been shown to have 30-40% amino acid identity with a human placental protein phosphotyrosine phosphatase, and CD45 isolated from human spleen was found to exhibit intrinsic protein phosphotyrosine phosphatase (EC 3.1.3.48) activity. In the present studies, we demonstrate that each of the known isoforms of murine CD45 has an equivalent basal level of protein phosphotyrosine phosphatase activity and establish that this enzymatic activity is associated with the cytoplasmic domain of the glycoprotein. Studies with three independent sets of well-characterized parental CD45+, mutant CD45-, and revertant CD45+ lymphoma cell lines indicate that loss of CD45 increases the phosphorylation of the src-related leukocyte-specific tyrosine protein kinase p56lck on tyrosine-505, a putative negative regulatory site. This suggests that CD45 may play a role in leukocyte growth regulation by altering the kinase activity of p56lck.

Animals↗
PubMed1988

Characterization of the human transferrin receptor produced in a baculovirus expression system.

Recombinant human transferrin receptor has been produced in a baculovirus expression system. Magnetic particles coated with an anti-transferrin receptor monoclonal antibody were used to immunoselect virus-infected Sf9 insect cells expressing the human transferrin receptor on their cell surface. Recombinant virus containing the human transferrin receptor cDNA was then plaque-purified from these cells. Biosynthetic labeling studies of infected cells showed that the human transferrin receptor is one of the major proteins made 2-3 days postinfection. The recombinant receptor made in insect cells is glycosylated and is also posttranslationally modified by the addition of a fatty acid moiety. However, studies with tunicamycin and endoglycosidases H and F showed that the oligosaccharides displayed on the recombinant receptor differ from those found on the naturally occurring receptor in human cells. As a consequence, the human receptor produced in the baculovirus system has an Mr of 82,000 and is smaller in size than the authentic receptor. About 30% of human transferrin receptors made in insect cells do not form intermolecular disulfide bonds, but are recognized by the anti-transferrin receptor antibody, B3/25, and bind specifically to a human transferrin-Sepharose column. Binding studies using 125I-labeled human transferrin showed that insect cells infected with the recombinant virus expressed an average of 5.8 +/- 0.9 X 10(5) transferrin receptors (Kd = 63 +/- 9 nM) on their cell surface. Thus, the human transferrin receptor produced in insect cells is biologically active and appears suitable for structural and functional studies.

Acylation↗
PubMed1987

Effects of monoclonal antibodies that block transferrin receptor function on the in vivo growth of a syngeneic murine leukemia.

The ability of monoclonal antibodies (MAbs) against the murine transferrin receptor to inhibit the growth of transplanted syngeneic AKR/J SL-2 leukemic cells has been investigated. Two rat IgM antibodies, RI7 208 and REM 17.2, which both block transferrin receptor function, inhibited the growth of SL-2 leukemic cells in vitro at concentrations of 5-10 micrograms per ml. However, RI7 208 was more effective than REM 17.2 in prolonging survival of tumor-bearing mice. The antitumor effects of RI7 208 MAb were dependent on both the antibody dose and number of leukemic cells inoculated. The serum clearance of [75Se]methionine-labeled RI7 208 and REM 17.2 antibodies was similar and consisted of an initial rapid phase over the first 2 days followed by a slower phase. A single dose of 2 mg of antibody maintained a serum MAb concentration (greater than 10 micrograms/ml) sufficient to inhibit SL-2 leukemic cell growth in vitro for 2-3 days. The liver, kidney, and spleen were the major sites at which each of the antibodies accumulated regardless of whether trace or saturating amounts of antibody were administered. The specific activity of antibody found in s.c. SL-2 tumors was about 2-fold less than that of liver. It was shown that multiple doses of R17 208 MAb administered on a schedule aimed at maintaining a therapeutic serum level of MAb for 1-3 weeks were more effective than a single dose. Further, administration of RI7 208 MAb, in combination with the anti-Thy-1.1 MAb 19E12, was more effective than either antibody alone. SL-2 mutant cells were selected that were resistant to growth inhibitory effects of RI7 208 in vitro. The effects of RI7 208 MAb on the growth of these mutant cells in vivo suggests the major mechanism by which the MAb inhibits SL-2 tumor growth is by directly blocking receptor function. Acute toxicity associated with administration of the MAb was minimal. However, assays of myeloid and erythroid colony-forming units in bone marrow and spleen of mice given multiple doses of RI7 208 showed a depression of stem cell activity in bone marrow and elevated numbers of erythroid and cellular colony-forming units in the spleen.

Animals↗
PubMed1987

Changes in gene expression induced by a phorbol diester: expression of IL 2 receptor, T3, and T cell antigen receptor.

Phorbol esters cause an apparent differentiation of human T leukemic cell lines. It was shown previously that TPA induces the expression of the interleukin 2 (IL 2) receptor and the T3 complex on some T cell lines, including CCRF-CEM. We demonstrate that expression of the IL 2 receptor correlated with an induction of the 3.5 and 1.5 kb IL 2 receptor mRNA. In addition, the TPA-induced expression of the T3 polypeptides was found to be accompanied by induction of a putative T cell antigen receptor heterodimer on CEM cells. This was demonstrated by the co-precipitation of the T cell receptor with T3 from digitonin-solubilized cells. The cells expressed high levels of T3 delta- and T cell receptor beta-chain mRNA in the absence of TPA. The effect of TPA was to cause a rapid accumulation of T cell receptor alpha-chain mRNA. This suggested that the alpha-chain gene was rearranged before TPA induction and that expression of the T cell receptor/T3 complex on the cell surface was regulated by the level of alpha-chain expression. It was also shown that cloned sublines of CEM cells which expressed different T cell antigen phenotypes differed in their response to TPA.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Structural variants of human T200 glycoprotein (leukocyte-common antigen).

Structural variation in the primary structure of human T200 glycoprotein has been detected. Three cDNA variants have been characterized each of which encode T200 molecules that differ in size as a result of sequence differences in their amino-terminal regions. The largest form of the molecule is distinguished from the smallest by an insert of 161 amino acids, after the first eight amino-terminal residues. The other variant has an insert at the same location of 47 amino acids identical to residues 75-121 in the larger insert. Both extra domains are rich in serine and threonine residues and are likely to display multiple O-linked oligosaccharides. These structural variants which probably arise by cell-type-specific alternative splicing provide a molecular basis for the previously observed structural and antigenic heterogeneity of T200 glycoprotein. In addition to the variable amino-terminal region, the external domain of human T200 glycoprotein consists of a second cysteine-rich region of about 400 amino acids, a single transmembrane-spanning region and a large cytoplasmic domain of 707 amino acids shared by all of the structural variants and highly conserved between species. The gene encoding human T200 is located on the long arm of chromosome 1.

Amino Acid Sequence↗
PubMed1987

Identification of the intermolecular disulfide bonds of the human transferrin receptor and its lipid-attachment site.

Structural studies of the human transferrin receptor have shown that the molecule is a disulfide-bonded dimer consisting of two identical subunits (Mr = 95,000) which are post-translationally modified by the addition of a fatty acyl moiety. Oligonucleotide site-directed mutagenesis has been used to obtain mutant molecules in which each of the four cysteines, residues 62, 67, 89 and 98, clustered within or adjacent to the membrane-spanning region were modified to serine. By first preparing mutants with only one of these cysteine residues modified to serine and then obtaining additional mutants in which different combinations of two cysteine residues were modified, we have shown that both cysteine 89 and cysteine 98, which are located in the extracellular domain of the receptor, are involved in intermolecular disulfide bonds. Further, we have identified cysteine 62 as the major site of acylation. Each of the mutant molecules is synthesized and transported to the cell surface when the modified human transferrin receptor cDNAs are transiently expressed in simian Cos cells. It should therefore now be possible to design experiments to determine whether these modified receptors bind transferrin normally and mediate iron uptake.

Acylation↗
PubMed1987

B-cell variant of mouse T200 (Ly-5): evidence for alternative mRNA splicing.

The complete cDNA sequence of mouse T200 glycoprotein from the pre-B-cell line 70Z/3 has been determined. The deduced protein sequence differs from that previously reported for a T-cell form of the molecule [Saga, Y., Tung, J.-S., Shen, F.-W. & Boyse, E. A. (1986) Proc. Natl. Acad. Sci. USA 83, 6940-6944] by the insertion of 139 amino acid residues in the amino-terminal region of the molecule. RNA transfer blotting using a cDNA probe encoding this sequence established that the predominant T200 mRNA species from B cells and cytotoxic T-cell clones but not T-helper cell clones or thymocytes contain all or part of the insert. Overlapping genomic clones of murine T200 were isolated and analysis of the intron-exon structure at the 5' end of the gene provides evidence that variants of T200 glycoprotein are generated by alternative mRNA splicing.

Amino Acid Sequence↗
PubMed1986

Evidence for two extracellular domains in the human interleukin-2 receptor: localization of IL-2 binding.

The human interleukin-2 (IL-2) receptor was quantitatively cleaved into two large disulfide-bonded fragments by either trypsin or endoproteinase lys-C (endo lys-C). The smaller fragment contains both N-linked oligosaccharides found in the intact receptor and is derived from the amino terminus of the molecule. The larger proteolytic fragment was metabolically labeled with 32PO4 and represents the carboxy terminus. The predicted cleavage sites of both enzymes lie in the region of the molecule encoded by exon 3. This pattern of limited proteolysis provides biochemical evidence that the extracellular region of the receptor is organized into two domains. This supports a structural model of the receptor in which the regions of internal homology encoded by exons 2 and 4 form independent disulfide-bonded domains connected by a hydrophilic segment. To determine the role of these domains in IL-2 binding, [125I]IL-2 was chemically cross-linked to the proteolytically cleaved receptor on the cell surface. The 125I-labeled complex obtained displayed N-linked oligosaccharides and had an Mr consistent with one molecule of IL-2 cross-linked to the smaller proteolytic fragment of the receptor. Thus, the amino-terminal domain of the IL-2 receptor appears to form an integral part of the IL-2 binding site.

Cell Line↗