CRK protein binds to two guanine nucleotide-releasing proteins for the Ras family and modulates nerve growth factor-induced activation of Ras in PC12 cells

1994 ◽  
Vol 14 (8) ◽  
pp. 5495-5500
Author(s):  
M Matsuda ◽  
Y Hashimoto ◽  
K Muroya ◽  
H Hasegawa ◽  
T Kurata ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Pc12 Cells ◽  
Sh2 Domain ◽  
Single Amino Acid ◽  
Guanine Nucleotide ◽  
Nerve Growth ◽  
Tyrosine Residues ◽  
Ngf Receptor ◽  
Ras Family

It has been reported that growth factors activate Ras through a complex of an adaptor type SH2-containing molecule, Grb2, and a Ras guanine nucleotide-releasing protein (GNRP), mSos. We report on the involvement of another adaptor molecule, CRK, in the activation of Ras. Overexpression of wild-type CRK proteins CRK-I and CRK-II enhanced the nerve growth factor (NGF)-induced activation of Ras in PC12 cells, although the basal level of GTP-bound active Ras was not altered. In contrast, mutants with a single amino acid substitution in either the SH2 or SH3 domain of the CRK-I protein inhibited the NGF-induced activation of Ras. Two GNRPs for the Ras family, mSos and C3G, were coimmunoprecipitated with the endogenous Crk proteins in PC12 cells. The association between C3G and the CRK mutants was dependent upon the presence of intact SH3. The SH2 domain of CRK bound to the SHC protein phosphorylated on tyrosine residues by NGF stimulation. The results demonstrate that, in addition to Grb2, CRK participates in signaling from the NGF receptor and that two GNRPs appear to transmit signals from these adaptor molecules to Ras.

scholarly journals CRK protein binds to two guanine nucleotide-releasing proteins for the Ras family and modulates nerve growth factor-induced activation of Ras in PC12 cells.

1994 ◽  
Vol 14 (8) ◽  
pp. 5495-5500 ◽  
Author(s):  
M Matsuda ◽  
Y Hashimoto ◽  
K Muroya ◽  
H Hasegawa ◽  
T Kurata ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Pc12 Cells ◽  
Sh2 Domain ◽  
Single Amino Acid ◽  
Guanine Nucleotide ◽  
Nerve Growth ◽  
Tyrosine Residues ◽  
Ngf Receptor ◽  
Ras Family

It has been reported that growth factors activate Ras through a complex of an adaptor type SH2-containing molecule, Grb2, and a Ras guanine nucleotide-releasing protein (GNRP), mSos. We report on the involvement of another adaptor molecule, CRK, in the activation of Ras. Overexpression of wild-type CRK proteins CRK-I and CRK-II enhanced the nerve growth factor (NGF)-induced activation of Ras in PC12 cells, although the basal level of GTP-bound active Ras was not altered. In contrast, mutants with a single amino acid substitution in either the SH2 or SH3 domain of the CRK-I protein inhibited the NGF-induced activation of Ras. Two GNRPs for the Ras family, mSos and C3G, were coimmunoprecipitated with the endogenous Crk proteins in PC12 cells. The association between C3G and the CRK mutants was dependent upon the presence of intact SH3. The SH2 domain of CRK bound to the SHC protein phosphorylated on tyrosine residues by NGF stimulation. The results demonstrate that, in addition to Grb2, CRK participates in signaling from the NGF receptor and that two GNRPs appear to transmit signals from these adaptor molecules to Ras.

Molecular cloning of the mouse grb2 gene: differential interaction of the Grb2 adaptor protein with epidermal growth factor and nerve growth factor receptors

1993 ◽  
Vol 13 (9) ◽  
pp. 5500-5512
Author(s):  
K L Suen ◽  
X R Bustelo ◽  
T Pawson ◽  
M Barbacid
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Pc12 Cells ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Binding Studies ◽  
Trk Receptors ◽  
Nerve Growth ◽  
Epidermal Growth

We report the isolation and molecular characterization of the mouse grb2 gene. The product of this gene, the Grb2 protein, is highly related to the Caenorhabditis elegans sem-5 gene product and the human GRB2 protein and displays the same SH3-SH2-SH3 structural motifs. In situ hybridization studies revealed that the mouse grb2 gene is widely expressed throughout embryonic development (E9.5 to P0). However, grb2 transcripts are not uniformly distributed, and in certain tissues (e.g., thymus) they appear to be regulated during development. Recent genetic and biochemical evidence has implicated the Grb2 protein in the signaling pathways that link cell surface tyrosine kinase receptors with Ras. We have investigated the association of the Grb2 protein with epidermal growth factor (EGF) and nerve growth factor (NGF) receptors in PC12 pheochromocytoma cells. EGF treatment of PC12 cells results in the rapid association of Grb2 with the activated EGF receptors, an interaction mediated by the Grb2 SH2 domain. However, Grb2 does not bind to NGF-activated Trk receptors. Mitogenic signaling of NGF in NIH 3T3 cells ectopically expressing Trk receptors also takes place without detectable association between Grb2 and Trk. These results suggest that whereas EGF and NGF can activate the Ras signaling pathway in PC12 cells, only the EGF receptor is likely to do so through a direct interaction with Grb2. Finally, binding studies with glutathione S-transferase fusion proteins indicate that Grb2 binds two distinct subsets of proteins which are individually recognized by its SH2 and SH3 domains. These observations add further support to the concept that Grb2 is a modular adaptor protein.

scholarly journals Alteration of binding properties and cytoskeletal attachment of nerve growth factor receptors in PC12 cells by wheat germ agglutinin.

1982 ◽  
Vol 94 (3) ◽  
pp. 710-717 ◽  
Author(s):  
R D Vale ◽  
E M Shooter
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Pc12 Cells ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Binding Properties ◽  
Nerve Growth ◽  
Total Binding ◽  
Ngf Receptor ◽  
Triton X

Incubation of PC12 cells preloaded with 125I-nerve growth factor (NGF) reveals rapidly and slowly dissociating binding components indicative of a heterogeneous population of receptors. If the cells are previously exposed to wheat germ agglutinin (WGA) for 30 min, NGF now binds to an apparently homogeneous receptor population which exhibit slow dissociation kinetics. Total binding is also reduced by 50%. If WGA is added subsequent to 125I-NGF, total binding is not diminished, but rapidly dissociating receptors occupied with NGF are all converted to the slowly dissociating form. This conversion of receptors occurs rapidly, reaching completion within 2 min at 37 degrees or 4 degrees C, and is unaffected by metabolic energy poisons, suggesting that WGA-induced slowly dissociating receptors are not the product of internalization. The effects of the lectin are blocked by the sugar N-acetyl-D-glucosamine, and the lectin-induced slowly dissociating receptors are converted back to rapidly dissociating receptors by addition of this same sugar. WGA also affects the association of the NGF receptor with the Triton X-100 cytoskeleton. Greater than 90% of bound 125I-NGF becomes associated with Triton X-100 insoluble cytoskeletons in the presence of the lectin, compared with less than 20% before lectin addition. Cytoskeleton association of the NGF receptor by WGA shows similar kinetics as the conversion of rapidly to slowly dissociating receptors. This interaction may be involved in the alteration of NGF-receptor binding properties produced by this lectin.

scholarly journals Molecular cloning of the mouse grb2 gene: differential interaction of the Grb2 adaptor protein with epidermal growth factor and nerve growth factor receptors.

1993 ◽  
Vol 13 (9) ◽  
pp. 5500-5512 ◽  
Author(s):  
K L Suen ◽  
X R Bustelo ◽  
T Pawson ◽  
M Barbacid
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Pc12 Cells ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Binding Studies ◽  
Trk Receptors ◽  
Nerve Growth ◽  
Epidermal Growth

We report the isolation and molecular characterization of the mouse grb2 gene. The product of this gene, the Grb2 protein, is highly related to the Caenorhabditis elegans sem-5 gene product and the human GRB2 protein and displays the same SH3-SH2-SH3 structural motifs. In situ hybridization studies revealed that the mouse grb2 gene is widely expressed throughout embryonic development (E9.5 to P0). However, grb2 transcripts are not uniformly distributed, and in certain tissues (e.g., thymus) they appear to be regulated during development. Recent genetic and biochemical evidence has implicated the Grb2 protein in the signaling pathways that link cell surface tyrosine kinase receptors with Ras. We have investigated the association of the Grb2 protein with epidermal growth factor (EGF) and nerve growth factor (NGF) receptors in PC12 pheochromocytoma cells. EGF treatment of PC12 cells results in the rapid association of Grb2 with the activated EGF receptors, an interaction mediated by the Grb2 SH2 domain. However, Grb2 does not bind to NGF-activated Trk receptors. Mitogenic signaling of NGF in NIH 3T3 cells ectopically expressing Trk receptors also takes place without detectable association between Grb2 and Trk. These results suggest that whereas EGF and NGF can activate the Ras signaling pathway in PC12 cells, only the EGF receptor is likely to do so through a direct interaction with Grb2. Finally, binding studies with glutathione S-transferase fusion proteins indicate that Grb2 binds two distinct subsets of proteins which are individually recognized by its SH2 and SH3 domains. These observations add further support to the concept that Grb2 is a modular adaptor protein.

scholarly journals PC12 cell mutants that possess low- but not high-affinity nerve growth factor receptors neither respond to nor internalize nerve growth factor.

1986 ◽  
Vol 102 (3) ◽  
pp. 830-843 ◽  
Author(s):  
S H Green ◽  
R E Rydel ◽  
J L Connolly ◽  
L A Greene
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Cell Surface ◽  
Pc12 Cells ◽  
Selection Procedure ◽  
Division Rate ◽  
Nerve Growth ◽  
High Affinity ◽  
Ngf Receptor ◽  
Epidermal Growth

Four mutant PC12 pheochromocytoma cell lines that are nerve growth factor (NGF)-nonresponsive (PC12nnr) have been selected from chemically mutagenized cultures by a double selection procedure: failure both to grow neurites in the presence of NGF and to survive in NGF-supplemented serum-free medium. The PC12nnr cells were deficient in all additional NGF responses surveyed: abatement of cell proliferation, changes in glycoprotein composition, induction of ornithine decarboxylase, rapid changes in protein phosphorylation, and cell surface ruffling. However, PC12nnr cells closely resembled non-NGF-treated PC12 cells in most properties tested: cell size and shape; division rate; protein, phosphoprotein, and glycoprotein composition; and cell surface morphology. All four PC12nnr lines differed from PC12 cells in three ways in addition to failure of NGF response: PC12nnr cells failed to internalize bound NGF by the normal, saturable, high-affinity mechanism present in PC12 cells. The PC12nnr cells bound NGF but entirely, or nearly entirely, at low-affinity sites only, whereas PC12 cells possess both high- and low-affinity NGF binding sites. The responses to dibutyryl cyclic AMP that were tested appeared to be enhanced or altered in the PC12nnr cells compared to PC12 cells. Internalization of, and responses to, epidermal growth factor were normal in the PC12nnr cells ruling out a generalized defect in hormonal binding, uptake, or response mechanisms. These findings are consistent with a causal association between the presence of high-affinity NGF receptors and of NGF responsiveness and internalization. A possible relationship is also suggested between regulation of cAMP responses and regulation of NGF responses or NGF receptor affinity.

Nerve growth factor stimulates rapid metabolic responses in PC12 cells

1995 ◽  
Vol 268 (4) ◽  
pp. C936-C943 ◽  
Author(s):  
S. Pitchford ◽  
K. De Moor ◽  
B. S. Glaeser
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Pc12 Cells ◽  
Protein Tyrosine Kinase ◽  
Metabolic Response ◽  
Nerve Growth ◽  
Biosensor System ◽  
Ngf Receptor ◽  
Silicon Based ◽  
Growth Factor Treatment

Research into the effects of nerve growth factor (NGF) has involved study of either the signal transduction process or the morphological result of growth factor treatment (cell proliferation and/or differentiation). The Cytosensor Microphysiometer, a silicon-based biosensor system that allows the continuous and real-time monitoring of extracellular acidification rate changes of cells, was used to study the response of PC12 cells to NGF. Stimulation resulted in a rapid increase in the acidification rate of cells in a concentration-dependent fashion (0.1-200 ng/ml NGF; mean effective concentration value of 153 +/- 54 pM). Inhibition of the NGF receptor-linked protein tyrosine kinase by either genistein or K252a attenuated the acidification rate response to NGF. In addition, the acidification response to NGF could be modified by inhibiting Na+/H+ exchange and, separately, glycolysis. This implicates these processes in the metabolic response of PC12 cells to NGF stimulation.

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