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B L Hempstead

Publications and source records attributed to B L Hempstead.

At least 37 records · Page 2Linked to original sources
PubMed1995

v-Crk modulation of growth factor-induced PC12 cell differentiation involves the Src homology 2 domain of v-Crk and sustained activation of the Ras/mitogen-activated protein kinase pathway.

Nerve growth factor (NGF) and epidermal growth factor (EGF) elicit contrasting actions on PC12 pheochromocytoma cells; NGF causes neuronal differentiation, and EGF induces proliferation. However, ectopic expression of the Src homology 2 (SH2) and SH3-containing oncogenic adaptor protein v-Crk in PC12 cells results in EGF-inducible neuronal differentiation (Hempstead, B. L., Birge, R. B., Fajardo, J. E., Glassman, R., Mahadeo, D., Kraemer, R., and Hanafusa, H. (1994) Mol. Cell. Biol. 14, 1964-1971). Here we show that v-Crk complexes with both the tyrosine-phosphorylated EGF receptor and the Ras guanine nucleotide exchange factor SOS in PC12 cells and is involved in an pathway analogous to that of Grb2. Expression of v-Crk results in an enhanced and sustained activation of Ras and mitogen-activated protein (MAP) kinase following EGF or NGF stimulation, implying that v-Crk can couple divergent tyrosine kinase pathways to Ras. To investigate the causal relationship between EGF receptor binding, MAP kinase activation, and neurite outgrowth, we stably expressed two v-Crk SH2 point mutants, v-Crk(R273N) and v-Crk(H294R) in PC12 cells. Mutations within the SH2 domain of v-Crk block binding of v-Crk to the tyrosine phosphorylated EGF receptor, compromise v-Crk's ability to cause EGF-dependent neurite outgrowth, and act in a dominant negative manner for NGF-induced neurite outgrowth. However, the kinetics of MAP kinase activation in EGF- or NGF-treated v-Crk-(R273N)PC12 cells was comparable with that in v-CrkPC12 cells. These data are consistent with a model in which v-Crk regulates the strength of a tyrosine kinase signal leading to prolonged activation of Ras and MAP kinase. However, the experiments with the SH2 mutants suggest that sustained activation, by itself, may not be sufficient to switch the fate of v-CrkPC12 cells from proliferation toward differentiation.

Amino Acid Sequence↗
PubMed1995

Expression of trk receptors in the developing and adult human central and peripheral nervous system.

A family of tyrosine receptor kinases known collectively as trk receptors plays an essential role in signal transduction mediated by nerve growth factor and related neurotrophins. To localize the major trk receptors (trkA, B and C) in the developing and adult central (CNS) and peripheral (PNS) nervous system, we generated monoclonal antibodies (MAbs) to extracellular (MAbs E7, E13, E16, E21, E29) and intracellular (MAb I2) domains of human trkA fused to glutathione S-transferase. Several MAbs (E7, E13, E16) recognized glycosylated trkA (gp140trk and gp110trk) in Western blots, one MAb (E7) recognized non-glycosylated (p80trk) and glycosylated trkA in immunoprecipitation assays, and two MAbs (E13, E29) detected trkA on the cell surface of NIH3T3 cells transfected with a trkA cDNA. Although generated to trkA fusion proteins, this panel of MAbs also recognized trkB and trkC in flow cytometric studies of NIH3T3 cells transfected with trkB or trkC cDNAs. Thus, we used these pan-trk MAbs to probe selected regions of the CNS and PNS including the hippocampus, nucleus basalis of Meynert, cerebellum, spinal cord, and dorsal root ganglion (DRG) to localize trkA, B, and C receptors in the developing and adult human nervous system. These studies showed that trk receptors are expressed primarily by neurons and are detectable very early in the developing hippocampus, cerebellum, spinal cord, and DRG. Although the distribution and intensity of trk immunoreactivity changed with the progressive maturation of the CNS and PNS, immunoreactive trk receptors were detected in neurons of the adult human hippocampus, nucleus basalis of Meynert, cerebellum, spinal cord, and DRG. This first study of trk receptor proteins in the developing and adult human CNS and PNS documents the expression of these receptors in subsets of neurons throughout the developing and adult nervous system. Thus, although the expression of trk receptor proteins is developmentally regulated, the constitutive expression of these neurotrophin receptors by neurons in many regions of the adult human CNS and PNS implies that mature trk receptor-bearing neurons retain the ability to respond to neurotrophins long after terminal neuronal differentiation is complete.

3T3 Cells↗
PubMed1995

NGF binding to the trk tyrosine kinase receptor requires the extracellular immunoglobulin-like domains.

Neurotrophins initiate their biological effects by activating members of the trk tyrosine kinase subfamily. The extracellular region of trk receptors is distinguished by several common structural features, including leucine-rich repeats, clusters of cysteine-rich domains, and two immunoglobulin-like domains. However, the receptor sequences required for ligand binding have not been localized. In order to define the domains involved in NGF binding, a series of chimeric receptors was constructed using cDNA sequences from rat trkA and trkB. The chimeric constructs were expressed after transient transfection in 293 cells and the expression of each receptor was verified by immunoprecipitation and immunoblot analysis. Equilibrium binding of transfected cells revealed that the two IgG domains of trkA are essential for NGF binding. The requirement for the two IgG domains was further confirmed by Scatchard analysis and affinity crosslinking with 125I-NGF. These results indicate that NGF binding is crucially dependent upon interactions with the IgG domains of the trkA receptor.

Base Sequence↗
PubMed1995

p75 and Trk: a two-receptor system.

The neurotrophin family of survival factors is distinguished by a unique receptor-signaling system that is composed of two transmembrane receptor proteins. Nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3 (NT-3) and NT-4/5 share similar protein structures and biological functions and interact with two different types of cell-surface proteins, the Trk family of receptor tyrosine kinases, and the p75, or low-affinity neurotrophin receptor. An important question is whether a dual receptor system is necessary for neurotrophin action. Evidence indicates that co-expression of the two genes for the p75 receptor and the Trk NGF receptor can potentially lead to greater responsiveness to NGF, and suggests additional levels of regulation for the family of neurotrophin factors.

Animals↗
PubMed1994

High affinity nerve growth factor binding displays a faster rate of association than p140trk binding. Implications for multi-subunit polypeptide receptors.

Nerve growth factor (NGF) binds to two cell surface receptors, p140trk and p75NGFR, which are both expressed in responsive sensory, sympathetic, and basal forebrain cholinergic neurons. While p140trk belongs to the family of receptor tyrosine kinases, p75NGFR is a member of the TNF/Fas/CD40/CD30 family of receptors. Current views of neurotrophin receptor function have tended to interpret p140trk as the high affinity NGF-binding site. To assess if the binding of NGF to p140trk was distinguishable from binding to high affinity sites on neuronal cells, PC12 cell sublines were generated which expressed p140trk alone, or coexpressed both p140trk and p75NGFR. Kinetic analysis of 125I-NGF binding indicates that it has an unusually slow rate of association with p140trk (k + 1 = 8 x 10(5) M-1 s-1). When both p140trk and p75NGFR receptors are coexpressed, the rate of association of NGF is increased 25-fold to produce a higher affinity binding site. An increase in the rate of internalization was also observed. Since high affinity binding and internalization are believed to be prerequisite for the biological activities of NGF, these results suggest that the biological effects by NGF are derived from a novel kinetic binding site that requires the expression of both receptors. The implications of these results with respect to multisubunit polypeptide receptors are discussed.

Animals↗
PubMed1994

Expression of the v-crk oncogene product in PC12 cells results in rapid differentiation by both nerve growth factor- and epidermal growth factor-dependent pathways.

The transforming gene of the avian sarcoma virus CT10 encodes a fusion protein (p47gag-crk or v-Crk) containing viral Gag sequences fused to cellular sequences consisting primarily of Src homology regions 2 and 3 (SH2 and SH3 sequences). Here we report a novel function of v-Crk in the mammalian pheochromocytoma cell line, PC12, whereby stable expression of v-Crk induces accelerated differentiation, as assessed by induction of neurites following nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) treatment compared with the effect in native PC12 cells. Surprisingly, however, these cells also develop extensive neurite processes after epidermal growth factor (EGF) stimulation, an event which is not observed in native PC12 cells. Following EGF or NGF stimulation of the v-CrkPC12 cells, the v-Crk protein itself became tyrosine phosphorylated within 1 min. Moreover, in A431 cells or TrkA-PC12 cells, which overexpress EGF receptors and TrkA, respectively, a GST-CrkSH2 fusion protein was indeed capable of binding these receptors in a phosphotyrosine-dependent manner, suggesting that v-Crk can directly couple to receptor tyrosine kinase pathways in PC12 cells. In transformed fibroblasts, v-Crk binds to specific tyrosine-phosphorylated proteins of p130 and paxillin. Both of these proteins are also complexed to v-Crk in PC12 cells, as evidenced by their coprecipitation with v-Crk in detergent lysates, suggesting that common effector pathways may occur in both cell types. However, whereas PC12 cellular differentiation can occur solely by overexpression of the v-Src or oncogenic Ras proteins, that induced by v-Crk requires a growth factor stimulatory signal, possibility in a two-step process.

Animals↗
PubMed1993

Immunohistochemical localization of Trk receptor protein in pediatric small round blue cell tumors.

Expression of Trk protein has been documented by Northern analysis in neuroblastomas with good prognosis. To localize the expression of this protein at the cellular level within individual tumors, we adapted a recently characterized pan-Trk antibody for use in formalin fixed, paraffin-embedded tissue. We have examined a group of small round blue cell tumors occurring in children, including both high and low stage neuroblastomas, to assess the presence or absence of Trk expression and its cellular localization. Positive staining for Trk protein was observed in four of four low stage (good prognosis) neuroblastomas, five of five primitive neuroectodermal tumors/Ewing's sarcoma, five of five rhabdomyosarcomas, and no lymphomas. Within the neuroblastomas, expression of Trk protein was most striking in ganglion cells, in which positive cytoplasmic staining was demonstrated regardless of tumor stage. The latter observation may lend further insight into the pathobiology of this malignant childhood tumor.

Antibodies↗
PubMed1992

Overexpression of the trk tyrosine kinase rapidly accelerates nerve growth factor-induced differentiation.

To investigate the role of the gp140trk receptor tyrosine kinase in nerve growth factor (NGF)-induced differentiation, we have overexpressed gp140trk in the NGF-responsive PC12 cell line. Here we demonstrate that overexpression of gp140trk results in marked changes in NGF-induced differentiation. Whereas PC12 cells elaborated neurites after 2 days of continuous exposure to NGF, PC12 cells overexpressing gp140trk by 20-fold(trk-PC12) began this process within hours. Compared with wild-type PC12 cells, trk-PC12 exhibited an increase in both high and low affinity NGF-binding sites. Furthermore, trk-PC12 cells displayed an enhanced level of NGF-dependent gp140trk autophosphorylation, and this activity was sustained for many hours following ligand binding. The tyrosine phosphorylation or activity of several cellular proteins, such as PLC-gamma 1, PI-3 kinase, and Erk1 and the expression of the mRNA for the late response gene transin were also sustained as a consequence of gp140trk overexpression. The data indicate that overexpression of gp140trk in PC12 cells markedly accelerates NGF-induced differentiation pathways, possibly through the elevation of gp140trk tyrosine kinase activity.

Animals↗
PubMed1991

The low-affinity p75 nerve growth factor (NGF) receptor mediates NGF-induced tyrosine phosphorylation.

Protein tyrosine phosphorylation is a potential mechanism for initial signaling in PC12 cells during differentiation in response to nerve growth factor (NGF). NGF-induced tyrosine phosphorylation has been found to be initiated by the trk protooncogene, which participates in the formation of high-affinity NGF binding sites. In contrast to transfection of wild-type low-affinity p75 NGF receptors, transfection of p75NGFR with mutations in the cytoplasmic domain resulted in an inability of NGF to elicit tyrosine phosphorylation of intracellular substrates, indicating that p75NGFR is involved in initiating phosphorylation events by NGF. Even though the p75NGFR receptor does not possess any inherent tyrosine kinase activity, these experiments demonstrate that the p75NGFR has a potential role in NGF-induced tyrosine phosphorylation.

Adrenal Gland Neoplasms↗
PubMed1991

The trk proto-oncogene product: a signal transducing receptor for nerve growth factor.

The trk proto-oncogene encodes a 140-kilodalton, membrane-spanning protein tyrosine kinase (p140prototrk) that is expressed only in neural tissues. Nerve growth factor (NGF) stimulates phosphorylation of p140prototrk in neural cell lines and in embryonic dorsal root ganglia. Affinity cross-linking and equilibrium binding experiments with 125I-labeled NGF indicate that p140prototrk binds NGF specifically in cultured cells with a dissociation constant of 10(-9) molar. The identification of p140prototrk as an NGF receptor indicates that this protein participates in the primary signal transduction mechanism of NGF.

Animals↗
PubMed1991

High-affinity NGF binding requires coexpression of the trk proto-oncogene and the low-affinity NGF receptor.

Nerve growth factor (NGF) interacts with two different low-affinity receptors that can be distinguished by affinity crosslinking. Reconstitution experiments by membrane fusion and transient transfection into heterologous cells indicate that high-affinity NGF binding requires coexpression and binding to both the low-affinity NGF receptor and the tyrosine kinase trk gene product. These studies reveal a new growth factor receptor-mediated mechanism of cellular differentiation involving trk and the low-affinity NGF receptor.

Animals↗
PubMed1991

Differential expression of MAG isoforms during development.

The myelin-associated glycoproteins (MAG) mediate the cell interactions of oligodendrocytes and Schwann cells with axons that are myelinated. MAG exists in two developmentally regulated isoforms: large MAG (L-MAG) and small MAG (S-MAG). In this paper, we have studied the tissue-specific and developmentally regulated alternative splicing of these isoforms using monospecific antibodies that recognize epitopes common to both isoforms or that are present only on L-MAG. In the central nervous system (CNS), L-MAG is the major form synthesized early in development, and it persists as a significant proportion of the MAG present in the adult. In the peripheral nervous system (PNS), L-MAG is expressed at modest levels during development; it is virtually absent in the adult. Thus, the expression of L-MAG is not limited to the CNS, as was formerly believed, suggesting that it plays a common role during the early stages of myelin formation by both oligodendrocytes and Schwann cells. In both the CNS and PNS, S-MAG is the predominant isoform in the adult. A higher-molecular-weight form of MAG is present in the PNS at low abundance, that is developmentally regulated, and appears to be a glycosylation variant. An analysis of the carbohydrate residues on MAG demonstrates that it contains both N-linked and O-linked sugars that could be modulated during development. These results suggest a possible mechanism for the regulation of MAG function during myelinogenesis via the expression of alternative isoforms and carbohydrate modifications.

Animals↗
PubMed1990

Deletion of cytoplasmic sequences of the nerve growth factor receptor leads to loss of high affinity ligand binding.

The nerve growth factor (NGF) receptor is a glycosylated transmembrane protein present on the cell surface as both high and low affinity forms, but biological responsiveness requires interactions of NGF with the high affinity site. We have tested the effects of mutations in the intracellular domain of the receptor upon its cell surface expression and equilibrium binding of 125I-NGF. Although mutant receptors lacking the entire cytoplasmic domain are processed and expressed at the cell surface and are capable of binding to NGF, the absence of cytoplasmic sequences leads to a loss of high affinity binding and to a lack of an appropriate cross-linking pattern as assessed by N-hydroxysuccinimidyl 4-azidobenzoate photoaffinity cross-linking. These results, taken together with the highly conserved nature of these cytoplasmic sequences, implies that the interaction of the receptor with an accessory molecule is necessary to form the high affinity receptor.

Base Sequence↗
PubMed1989

Expression of functional nerve growth factor receptors after gene transfer.

Nerve growth factor (NGF) interacts with both high affinity (Kd = 10(-10)-10(-11)M) and low affinity (Kd = 10(-8)-10(-9)M) receptors; the binding of NGF to the high affinity receptor is correlated with biological actions of NGF. To determine whether a single NGF binding protein is common to both forms of the receptor, a full-length receptor cDNA was introduced in the NR18 cell line, an NGF receptor-deficient variant of the PC12 pheochromocytoma cell line. The transformant displayed (i) both high and low affinity receptors detectable by receptor binding; (ii) an affinity cross-linking pattern with 125I-labeled NGF similar to that of the parent PC12 cell line; and (iii) biological responsiveness to NGF as assayed by induction of c-fos transcription. These findings support the hypothesis that a single binding protein is common to both forms of the NGF receptor and suggest that an additional protein is required to produce the high affinity form of the NGF receptor.

Animals↗
PubMed1983

Selective phosphorylation of the IgE receptor in antigen-stimulated rat mast cells.

Purified rat serosal mast cells were sensitized with mouse immunoglobulin E (IgE) anti-2,4-dinitrophenyl antibody, partially depleted of phosphate, labeled with [32P]orthophosphate, and stimulated with dinitrophenylated bovine serum albumin or control protein. After 15-120 seconds at 37 degrees C, the cells were extracted with nonionic detergent. IgE receptors were purified by repetitive affinity chromatography and were analyzed by NaDodSO4/polyacrylamide gel electrophoresis and radioautography. Antigenic stimulation of intact rat mast cells produced a rapid and marked increase in the phosphorylation of the surface-exposed alpha component of the IgE receptor. However, phosphorylation of the 33,000 Mr beta component of the IgE receptor was not altered significantly by antigen stimulation. This suggests that the selective increase in phosphorylation of the IgE receptor alpha component may be part of the physiologic mediator secretion process triggered by antigen.

Animals↗
PubMed1981

The cell surface receptor for immunoglobulin E. Effect of tunicamycin on molecular properties of receptor from rat basophilic leukemia cells.

Cell surface receptors for immunoglobulin E were isolated by repetitive affinity chromatography from rat basophilic leukemia cells biosynthetically labeled with L-[35S]methionine and D-[3H]mannose. Native immunoglobulin E receptor appeared as a very broad band in the 45,000 to 62,000 Mr region in sodium dodecyl sulfate polyacrylamide gels. However, from cells cultured in the presence of tunicamycin, a relatively narrow band with an apparent Mr of 38,000 was isolated. The 38,000 Mr band rebound to immunoglobulin E-Sepharose, was immunoprecipitated with antibodies to immunoglobulin E receptor, shared tryptic peptides with native receptor, and was labeled with L-[35S]methionine but not D-[3H]mannose, and thus appears to be immunoglobulin E receptor lacking N-linked oligosaccharides. It is demonstrated that N-linked oligosaccharides account for much of the apparent heterogeneity of native receptor in sodium dodecyl sulfate polyacrylamide gels and in two-dimensional gel electrophoresis. A receptor-associated protein with apparent Mr = 30,000, prominently labeled with L-[35S]methionine but not with D-[3H]mannose, did not have altered molecular properties when isolated from tunicamycin-cultured cells, and did not share tryptic peptides with receptor.

Animals↗