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A Ullrich

Publications and source records attributed to A Ullrich.

At least 217 records · Page 12Linked to original sources
PubMed1992

Tyrosine phosphorylation of vav proto-oncogene product containing SH2 domain and transcription factor motifs.

Activation of receptor-linked and cytoplasmic protein tyrosine kinases is crucial in the control of normal and abnormal cell growth and differentiation. Some substrates of protein tyrosine kinases such as phospholipase C gamma and ras GTPase-activating protein (GAP) contain sequences homologous to the src protein domains SH2 and SH3 (refs 3-9). The proto-oncogene vav is expressed in haematopoietic cells and its product Vav contains sequence motifs commonly found in transcription factors, such as helix-loop-helix, leucine-zipper and zinc-finger motifs and nuclear localization signals, as well as a single SH2 and two SH3 domains. Here we show that stimulation of T-cell antigen receptor on normal human peripheral blood lymphocytes or on human leukaemic T cells, and the crosslinking of IgE receptors on rat basophilic leukaemia cells, both promote the phosphorylation of tyrosine residues in Vav. Moreover, activation of the receptor for epidermal growth factor leads to marked tyrosine phosphorylation of Vav in cells transiently expressing vav, and Vav associates with the receptor through its SH2 domain. We propose that vav encodes a new class of substrates whose tyrosine phosphorylation may provide a mechanism for direct signal transduction linking receptors at the cell surface to transcriptional control.

3T3 Cells↗
PubMed1992

Tyrosine phosphatase inhibition permits analysis of signal transduction complexes in p185HER2/neu-overexpressing human tumor cells.

The HER2/neu gene encodes a receptor tyrosine kinase that is highly homologous to the epidermal growth factor receptor. Overexpression of the receptor in mammary and ovarian carcinoma correlates with poor patient prognosis. To determine how the overexpression of a normal receptor leads to the generation of an oncogenic signal, we compared the patterns of tyrosine phosphorylation in tumor-derived human cell lines expressing high levels of p185HER2/neu. In intact SKBR3 cells, basal phosphorylation of p185HER2/neu was not detected. However, pretreatment of cells with the tyrosine phosphatase inhibitor, sodium orthovanadate, led to the detection of phosphotyrosine on phospholipase C-gamma (PLC-gamma), GTPase-activating protein but not on the RAF-1 kinase. Strikingly, PLC-gamma was detected in a complex which contained multiple tyrosine-phosphorylated polypeptides. This complex was detected only in cytoplasmic fractions and had a distinct composition in different p185HER2/neu-overexpressing cell lines. Although GTPase-activating protein has been found previously in association with proteins of 190 and 62 kDa in fibroblasts, in SKBR3 cells it was found associated with multiple additional tyrosine-phosphorylated polypeptides. These experiments show that SKBR3 cells possess high levels of protein tyrosine phosphatase that can act upon p185HER2/neu. Moreover, they reveal, for the first time, the presence of PLC-gamma and GTPase-activating protein in cytosolic complexes containing a variety of other tyrosine-phosphorylated polypeptides. These observations suggest novel possibilities for the specific definition of receptor-generated signals in tumor cells.

Amino Acid Sequence↗
PubMed1992

Expression of a type I insulin-like growth factor receptor with low affinity for insulin-like growth factor II.

We investigated the binding properties of the type I insulin-like growth factor (IGF) receptor expressed in NIH-3T3 fibroblasts transfected with a human type I receptor cDNA. Cell surface receptors bound IGF-I with KD = 1 nM as predicted. Although recent studies have suggested that IGF-I and IGF-II bind to type I receptors with near-equal affinity, the receptors in this system bound IGF-II with much lower affinity (KD = 15-20 nM). When type I receptors from the transfected cells were solubilized and immunopurified, however, both 125I-IGF-I and 125I-IGF-II bound to the purified receptors with extremely high and relatively similar affinities (KD = 8 and 17 pM respectively). Thus the immunopurified receptors had higher affinity but lower specificity for the two ligands. The monoclonal antibody alpha IR-3 effectively inhibited IGF-I binding to cell surface receptors (75 +/- 10%), but did not inhibit IGF-II binding. In the purified receptor assay, alpha IR-3 also inhibited IGF-I binding more effectively than IGF-II binding (38 +/- 7% versus 10 +/- 4%). We conclude that the products of this cDNA can account for the binding patterns that we previously observed in receptors immunopurified from human placenta. The differential effect of alpha IR-3 on IGF-I versus IGF-II raises the possibility that these homologous growth factors bind to immunologically distinct epitopes on the type I receptor.

3T3 Cells↗
PubMed1992

Immunoglobulin heavy chain-binding protein binds to misfolded mutant insulin receptors with mutations in the extracellular domain.

Cell-surface proteins are transported through the endoplasmic reticulum and Golgi apparatus en route to the plasma membrane. Previously, we have identified three point mutations in the insulin receptor gene that impair transport of the mutant receptors to the cell surface: Asn15----Lys, His209----Arg, and Phe382----Val. Furthermore, these mutations impair post-translational processing steps that normally occur as the receptors are transported through the endoplasmic reticulum and Golgi apparatus. In this study, we have demonstrated that the unprocessed Arg209 and Val382 mutant proreceptors are bound to the immunoglobulin heavy chain-binding protein (BiP) in the endoplasmic reticulum. This was demonstrated by the fact that monoclonal anti-BiP antibody coimmunoprecipitated the mutant proreceptors. Moreover, when ATP was added to the immunoprecipitates, the mutant proreceptors were released from BiP. In contrast, neither the normal human insulin receptor nor the Lys15 mutant proreceptor was coimmunoprecipitated by anti-BiP antibody. It seems likely that the Lys15 receptor also binds BiP, but that the affinity was too low to resist dissociation during the stringent washing of the immunoprecipitate. In conclusion, these observation are consistent with the hypothesis that binding to BiP explains the impaired transport of mutant receptors through the endoplasmic reticulum and Golgi apparatus to the plasma membrane.

3T3 Cells↗
PubMed1992

Effects of interferons on the expression of the proto-oncogene HER-2 in human ovarian carcinoma cells.

The over-expression of the proto-oncogene HER-2 (c-erbB-2/neu) in ovarian, endometrial and mammary carcinoma is an important indicator for poor prognosis. We have previously shown in 3 out of 4 ovarian carcinoma cell lines an interferon-gamma (IFN-gamma)-mediated reduction in HER-2 specific protein and RNA levels. The oncogene expression was lowered only in the ovarian carcinoma cell lines but not in 3 IFN-gamma-sensitive human breast cancer cell lines. We extended our observations also to IFN type I, alpha and omega. The expression of the oncogene was measured by both the p185HER-2 ELISA and in selected cases by a living cell radioimmunoassay using the monoclonal antibody (MAb) 4D5 against the extracellular domain. Both IFN types reduced the expression of HER-2 in the ovarian carcinoma cell lines OVCAR-3, HTB-77, 2774 and SKOV-6, and in the SKUT-2 endometrial carcinoma cells. In contrast, SKOV-8 human ovarian carcinoma cells were sensitive for both IFN types regarding proliferation, but only IFN-gamma reduced proto-oncogene expression. In the SKBR-3 human mammary carcinoma cells, neither IFN type had an effect on HER-2 expression. The antibodies 4D5, 7C2, 3E8, and 3H4 which bind to the extracellular domain of p185HER-2 protein specifically inhibited anchorage-independent growth of SKBR-3 and HTB-77 cells. Expression of the oncogene HER-2 is the leading prognostic factor in ovarian cancer. Its modulation might represent a mechanism by which IFNs inhibit cell proliferation.

Antibodies, Monoclonal↗
PubMed1992

SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma.

Several cytoplasmic tyrosine kinases contain a conserved, non-catalytic stretch of approximately 100 amino acids called the src homology 2 (SH2) domain, and a region of approximately 50 amino acids called the SH3 domain. SH2/SH3 domains are also found in several other proteins, including phospholipase C-gamma (PLC gamma). Recent studies indicate that SH2 domains promote association between autophosphorylated growth factor receptors such as the epidermal growth factor (EGF) receptor and signal transducing molecules such as PLC gamma. Because SH2 domains bind specifically to protein sequences containing phosphotyrosine, we examined their capacity to prevent tyrosine dephosphorylation of the EGF and other receptors with tyrosine kinase activity. For this purpose, various SH2/SH3 constructs of PLC gamma were expressed in Escherichia coli as glutathione-S-transferase fusion proteins. Our results show that purified SH2 domains of PLC gamma are able to prevent tyrosine dephosphorylation of the EGF receptor and other receptors with tyrosine activity. The inhibition of tyrosine dephosphorylation paralleled the capacity of various SH2-containing constructs to bind to the EGF receptor, suggesting that the tyrosine phosphatase and the SH2 domain compete for the same tyrosine phosphorylation sites in the carboxy-terminal tail of the EGF receptor. Analysis of the phosphorylation sites protected from dephosphorylation by PLC gamma-SH2 revealed substantial inhibition of dephosphorylation of Tyr992 at 1 microM SH2. This indicates that Tyr992 and its flanking sequence is the high-affinity binding site for SH2 domains of PLC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1992

The Xmrk receptor tyrosine kinase is activated in Xiphophorus malignant melanoma.

Xmrk encodes a putative transmembrane glycoprotein of the tyrosine kinase family and is a melanoma-inducing gene in Xiphophorus. We attempted to investigate the biological function of the putative Xmrk receptor by characterizing its signalling properties. Since a potential ligand for Xmrk has not yet been identified, it has been difficult to analyse the biochemical properties and biological function of this cell surface protein. In an approach towards such analyses, the Xmrk extracellular domain was replaced by the closely related ligand-binding domain sequences of the human epidermal growth factor receptor (HER) and the ligand-induced activity of the chimeric HER-Xmrk protein was examined. We show that the Xmrk protein is a functional receptor tyrosine kinase, is highly active in malignant melanoma and displays a constitutive autophosphorylation activity possibly due to an activating mutation in its extracellular or transmembrane domain. In the focus formation assay the HER-Xmrk chimera is a potent transforming protein equivalent to other tyrosine kinase oncoproteins.

Amino Acid Sequence↗
PubMed1992

Activation of insulin-epidermal growth factor (EGF) receptor chimerae regulates EGF receptor binding affinity.

Cell surface tyrosine kinase receptors are subject to a rapid activation by their ligand, which is followed by secondary regulatory processes. The IHE2 cell line is a unique model system to study the regulation of EGF binding to EGF receptors after activation of the EGF receptor kinase. IHE2 cells express both a chimeric insulin-EGF receptor kinase (IER) and a kinase-deficient EGF receptor (HER K721A). We have previously reported that IER is an insulin-responsive EGF receptor tyrosine kinase that activates one or several serine/threonine kinases, which in turn phosphorylate(s) the unoccupied HER K721A. In this article we show that insulin through IER activation induces a decrease in 125I-EGF binding to IHE2 cells. Scatchard analysis indicates that, as for TPA, the effect of insulin can be accounted for by a loss of the high affinity binding of EGF to HER K721A. Since this receptor transmodulation persists in protein kinase C downregulated IHE2 cells, it is likely to be due to a mechanism independent of protein kinase C activation. Using an in vitro system of 125I-EGF binding to transmodulated IHE2 membranes, we illustrate that the inhibition of EGF binding induced by IER activation is related to the phosphorylation state of HER K721A. Further, studies with phosphatase 2A, or at a temperature (4 degrees C) where only IER is functional, strongly suggest that the loss of high affinity EGF binding is related to the serine/threonine phosphorylation of HER K721A after IER activation. Our results provide evidence for a "homologous desensitization" of EGF receptor binding after activation of the EGF receptor kinase of the IER receptor.

Cell Line↗
PubMed1992

Kinetics of binding, endocytosis, and recycling of EGF receptor mutants.

This report describes analysis of factors which regulate the binding of EGF to EGF receptor, receptor internalization, and receptor recycling. Three different methods were used to inhibit high-affinity EGF binding as measured at equilibrium: treatment of cells with an active phorbol ester (PMA), binding of a mAb directed against the EGF receptor (mAb108), and truncation of most of the cytoplasmic domain of the receptor. These treatments reduced the rate at which low concentrations of EGF bound to cells, but did not affect the rate of EGF dissociation. We conclude that high-affinity EGF binding on living cells results from a difference in the apparent on rate of EGF binding. We then used these conditions and cell lines to test for the rate of EGF internalization at different concentrations of EGF. We demonstrate that internalization of the EGF receptor is stimulated roughly 50-fold at saturating concentrations of EGF, but is stimulated an additional two- to threefold at low concentrations (less than 1 nM). Four treatments reduce the rate of internalization of low concentrations of EGF to the rate seen at saturating EGF concentrations. Phorbol ester treatment and mAb108 binding to "wild type" receptor reduce this rate (and reduce high-affinity binding). Point mutation at Lys721 (kinase negative EGF receptor) and point mutation at Thr654 (removing a major site of protein kinase C phosphorylation) reduce the internalization rate, without affecting high-affinity binding. We suggest that while EGF stimulates endocytosis for all receptors, high-affinity receptors bind and are internalized more quickly than low-affinity receptors. Tyrosine kinase activity and the Thr654 region appear necessary for this response.

3T3 Cells↗
PubMed1992

Differential effects of carboxy-terminal sequence deletions on platelet-derived growth factor receptor signaling activities and interactions with cellular substrates.

Chimeric receptors composed of the human epidermal growth factor receptor (EGF-R) extracellular domain fused to wild-type and truncated platelet-derived growth factor receptor (PDGF-R) intracellular sequences were stably expressed in NIH 3T3 cells devoid of endogenous EGF-Rs. This experimental system allowed us to investigate the biological activity of PDGF-R cytoplasmic-domain mutants in PDGF-R-responsive NIH 3T3 cells by activating PDGF-specific signaling pathways with EGF. Deletion of 74 carboxy-terminal amino acids severely impaired the ability of the PDGF-R cytoplasmic domain to associate with cellular substrates in vitro. This deletion also inhibited receptor and substrate phosphorylation, reduced the receptor's mitogenic activity, and completely abolished its oncogenic signaling potential. Surprisingly, removal of only six additional amino acids, including Tyr-989, restored substantial receptor and substrate phosphorylation capacity as well as transforming potential and yielded a receptor with wild-type levels of ligand-induced mitogenic activity. However, the ability of this chimera to bind phospholipase C gamma was severely impaired in comparison with the ability of the wild-type receptor, while the association with other cellular proteins was not affected. Further deletion of 35 residues, including Tyr-977, nearly abolished all PDGF-R cytoplasmic-domain biological signaling activities. None of the three C-terminal truncations completely abolished the mitogenic potential of the receptors or had any influence on ligand binding or receptor down regulation. Together, these data implicate the 80 C-terminal-most residues of the PDGF-R, and possibly Tyr-989, in phospholipase C gamma binding, while receptor sequences upstream from Asp-988 appear to be essential for specific interactions with other cellular polypeptides such as ras GTPase-activating protein and phosphatidylinositol 3-kinase. Thus, the mutants described here allow the separation of distinct PDGF-activated signaling pathways and demonstrate that phospholipase C gamma phosphorylation is not required for mitogenesis and transformation.

3T3 Cells↗
PubMed1992

The SH2 and SH3 domain-containing Nck protein is oncogenic and a common target for phosphorylation by different surface receptors.

Signalling proteins such as phospholipase C-gamma (PLC-gamma) or GTPase-activating protein (GAP) of ras contain conserved regions of approximately 100 amino acids termed src homology 2 (SH2) domains. SH2 domains were shown to be responsible for mediating association between signalling proteins and tyrosine-phosphorylated proteins, including growth factor receptors. Nck is an ubiquitously expressed protein consisting exclusively of one SH2 and three SH3 domains. Here we show that epidermal growth factor or platelet-derived growth factor stimulation of intact human or murine cells leads to phosphorylation of Nck protein on tyrosine, serine, and threonine residues. Similar stimulation of Nck phosphorylation was detected upon activation of rat basophilic leukemia RBL-2H3 cells by cross-linking of the high-affinity immunoglobulin E receptors (Fc epsilon RI). Ligand-activated, tyrosine-autophosphorylated platelet-derived growth factor or epidermal growth factor receptors were coimmunoprecipitated with anti-Nck antibodies, and the association with either receptor molecule was mediated by the SH2 domain of Nck. Addition of phorbol ester was also able to stimulate Nck phosphorylation on serine residues. However, growth factor-induced serine/threonine phosphorylation of Nck was not mediated by protein kinase C. Interestingly, approximately fivefold overexpression of Nck in NIH 3T3 cells resulted in formation of oncogenic foci. These results show that Nck is an oncogenic protein and a common target for the action of different surface receptors. Nck probably functions as an adaptor protein which links surface receptors with tyrosine kinase activity to downstream signalling pathways involved in the control of cell proliferation.

3T3 Cells↗
PubMed1992

Anti-oncogenic activity of signalling-defective epidermal growth factor receptor mutants.

Overexpression and autocrine activation of the epidermal growth factor receptor (EGF-R) cause transformation of cultured cells and correlate with tumor progression in cancer patients. Dimerization and transphosphorylation are crucial events in the process by which receptors with tyrosine kinase activity generate normal and transforming cellular signals. Interruption of this process by inactive receptor mutants offers the potential to inhibit ligand-induced cellular responses. Using recombinant retroviruses, we have examined the effects of signalling-incompetent EGF-R mutants on the growth-promoting and transforming potential of ligand-activated, overexpressed wild-type EGF-R and the v-erbB oncogene product. Expression of a soluble extracellular EGF-R domain had little if any effect on the growth and transformation of NIH 3T3 cells by either tyrosine kinase. However, both a kinase-negative EGF-R point mutant (HERK721A) and an EGF-R lacking 533 C-terminal amino acids efficiently inhibited wild-type EGF-R-mediated, de novo DNA synthesis and cell transformation in a dose-dependent manner. Furthermore, coexpression with the v-erbBES4 oncogene product in NIH 3T3 cells resulted in transphosphorylation of the HERK721A mutant receptor and reduced soft-agar colony growth but had no effect in a focus formation assay. These results demonstrate that signalling-defective receptor tyrosine kinase mutants differentially interfere with oncogenic signals generated by either overexpressed EGF-R or the retroviral v-erbBES4 oncogene product.

3T3 Cells↗
PubMed1992

Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors.

One of the immediate cellular responses to stimulation by various growth factors is the activation of a phosphatidylinositol (PI) 3-kinase. We recently cloned the 85-kDa subunit of PI 3-kinase (p85) from a lambda gt11 expression library, using the tyrosine-phosphorylated carboxy terminus of the epidermal growth factor (EGF) receptor as a probe (E. Y. Skolnik, B. Margolis, M. Mohammadi, E. Lowenstein, R. Fischer, A. Drepps, A. Ullrich, and J. Schlessinger, Cell 65:83-90, 1991). In this study, we have examined the association of p85 with EGF and platelet-derived growth factor (PDGF) receptors and the tyrosine phosphorylation of p85 in 3T3 (HER14) cells in response to EGF and PDGF treatment. Treatment of cells with EGF or PDGF markedly increased the amount of p85 associated with EGF and PDGF receptors. Binding assays with glutathione S-transferase (GST) fusion proteins demonstrated that either Src homology region 2 (SH2) domain of p85 is sufficient for binding to EGF and PDGF receptors and that receptor tyrosine autophosphorylation is required for binding. Binding of a GST fusion protein expressing the N-terminal SH2 domain of p85 (GST-N-SH2) to EGF and PDGF receptors was half-maximally inhibited by 2 and 24 mM phosphotyrosine (P-Tyr), respectively, suggesting that the N-SH2 domain interacts more stably with PDGF receptors than with EGF receptors. The amount of receptor-p85 complex detected in HER14 cells treated with EGF or PDGF. Growth factor treatment also increased the amount of p85 found in anti-PDGF-treated HER14 cells, suggesting that the vast majority of p85 in the anti-P-Tyr fraction is receptor associated but not phosphorylated on tyrosine residues. Only upon transient overexpression of p85 and PDGF receptor did p85 become tyrosine phosphorylated. These are consistent with the hypothesis that p85 functions as an adaptor molecule that targets PI 3-kinase to activated growth factor receptors.

3T3 Cells↗
PubMed1992

Roles of insulinlike growth factor 1 (IGF-1) and the IGF-1 receptor in epidermal growth factor-stimulated growth of 3T3 cells.

BALB/c3T3 cells are exquisitely growth regulated and require platelet-derived growth factor, epidermal growth factor (EGF), and insulinlike growth factor 1 (IGF-1) for growth. When BALB/c3T3 cells are transfected with plasmids constitutively expressing both EGF and the human IGF-1 receptor mRNAs, the cells are capable of growing in serum-free medium without the addition of any exogenous growth factor. These cells, called p5 cells, can grow for prolonged periods in serum-free medium. BALB/c3T3 cells transfected with only the IGF-1 receptor expression plasmid (p6 cells) do not grow in serum-free medium but do grow if IGF-1 (or insulin in supraphysiological concentrations) is added. p6 cells also grow in response to EGF, confirming that the combination of EGF and an overexpressed IGF-1 receptor is sufficient for the growth of 3T3 cells. We have found that in EGF-stimulated p6 cells there is an increase in the expression of IGF-1 mRNA, that IGF-1 is secreted into the medium, and that the growth of p5 cells and EGF-stimulated p6 cells is inhibited by exposure to antisense oligodeoxynucleotides to IGF-1 receptor RNA. Finally, while cells constitutively expressing both EGF and EGF receptor RNAs grow, albeit modestly, in serum-free medium, their growth is also inhibited by an antisense oligodeoxynucleotide to IGF-1 receptor RNA. In contrast, in cells overexpressing the IGF-1 receptor, IGF-1-mediated cell growth occurs independently of the platelet-derived growth factor and EGF receptors (Z. Pietrzkowski, R. Lammers, G. Carpenter, A. M. Soderquist, M. Limardo, P. D. Phillips, A. Ullrich, and R. Baserga, Cell Growth Differ. 3:199-205, 1992, and this paper). These data indicate that an important role for EGF is participation in the activation of an autocrine loop based on the IGF-1-IGF-1 receptor interaction, which is obligatory for the proliferation of 3T3 cells.

3T3 Cells↗
PubMed1992

Evidence that two naturally occurring human insulin receptor alpha-subunit variants are immunologically distinct.

The IgG from a patient (Italy 2 [I2]) with hypoglycemia, due to autoantibodies to the insulin receptor, was purified on protein A Sepharose into two fractions that were tested in various human tissues and cells. The IgG fraction that bound protein A (absorbed IgG [IgGa]) nearly completely inhibited the binding of 125I-labeled insulin to various cells or tissues (placenta, IM-9, adipocytes, HEp-2-larynx cells, Epstein-Barr virus lymphocytes) but not greater than 50% of 125I-labeled insulin binding to human liver membranes. Conversely, both the IgG fraction from this patient, which did not bind protein A (flow-through IgG [IgGb]), and the IgGa fraction from a second similar patient (Italy 1 [I-1]) almost completely inhibited the binding of 125I-labeled insulin to liver membranes. The IgGa fraction from patient I-2 did not change receptor affinity because 50% inhibition of 125I-labeled insulin binding was not affected by either the presence or absence of these IgG fractions. Furthermore, liver binding data were not due to cross-reaction of 125I-labeled insulin to the insulinlike growth factor I receptor, and treatment of liver membranes with neuraminidase did not alter the inhibitory effect of the IgGa fraction from patient I-2 on 125I-labeled insulin binding to liver. Binding inhibition experiments performed with cells transfected with and overexpressing the -12 (human insulin receptor [HIR]-A) or the +12 (HIR-B) variant of HIR revealed that the IgGa fraction from patient I-2 inhibited 125I-labeled insulin binding to the HIR-A receptor but not to the HIR-B receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1992

Activation and regulation of the insulin receptor kinase.

For the insulin receptor and the EGF receptor it is believed that ligand occupancy results in interactions within the heterotetrameric alpha 2 beta 2 insulin receptor or between monomeric EGF receptors. These interactions then activate the intracellular receptor tyrosine kinase which induces receptor autophosphorylation and phosphorylation of cellular substrates. In the present study we have approached the nature of this receptor activation and autophosphorylation. We have investigated whether these phenomena occur via an intra--or an intermolecular process. To this end the following receptor model system consisting of two receptors was co-expressed in NIH 3T3 cells: a kinase inactive human insulin receptor (HIR K1018A) and a chimeric (EIR) receptor corresponding to the extracellular and transmembrane domains of the human EGF receptor and the cytosolic domain of the human insulin receptor beta subunit. Using this system we found that stimulation of the cells with EGF induced tyrosine autophosphorylation of the EGF-insulin receptor chimera (150 kd) and tyrosine phosphorylation of the beta-subunit of the kinase-deficient insulin receptor (95 kd). The phosphopeptides of the autophosphorylated cytoplasmic domain of the EGF-insulin receptor chimera were comparable to those of the transphosphorylated beta subunit of the kinase-deficient insulin receptor and the wild type human insulin receptor. When immunoaffinity purified EGF-insulin receptor hybrids and kinase-deficient insulin receptors were used in a cell lysate phosphorylation assay, it was found that addition of EGF produced [32P]-labeling of both receptor species. In conclusion, we have shown that tyrosine transphosphorylation can occur between homologous receptor domains. This transphosphorylation and transactivation could be a possible mechanism for signal amplification.2+ domain could influence interactions between the receptor and cellular structures and, as such, play a key role in signal transduction.

Animals↗