Biomedical subjects

Y Okabayashi

Publications and source records attributed to Y Okabayashi.

At least 19 recordsLinked to original sources

PubMed2001

Shc mediates ligand-induced internalization of epidermal growth factor receptors.

In order to clarify the physiological relevance of the interaction between Shc and adaptins, components of plasma membrane-coated pit adaptor complex AP2, we investigated the role of Shc in ligand-induced endocytosis of epidermal growth factor (EGF) receptors. In vitro peptide binding assay showed that alpha-adaptin bound to the wild-type peptide corresponding to amino acids 346-355 of Shc, RDLFDMKPFE, but not to the mutant peptide in which both phenylalanines at 349 and 354 were substituted for alanines (FA). Using adenovirus vectors carrying a herpes simplex virus epitope-tagged 52-kDa wild-type Shc and Shc FA, we examined the interaction between Shc, AP2, and EGF receptors in intact cells. Alpha-adaptin bound to wild-type Shc in an EGF-dependent manner, whereas EGF-dependent association of alpha-adaptin with Shc FA was markedly reduced. In addition, EGF increased the amount of alpha-adaptin coprecipitated with EGF receptors in cells expressing wild-type Shc but not Shc FA. These results suggest that EGF stimulates Shc-AP2 complex formation and association of Shc-AP2 complexes with EGF receptors. Internalization assay showed that (125)I-EGF internalization was reduced in cells overexpressing Shc FA. Immunofluorescence study showed that punctate staining along the plasma membrane border as well as punctate pattern characteristic of cytoplasmic vesicles near the plasma membrane was enhanced in cells expressing wild-type Shc. These results suggest, therefore, the implication of Shc in ligand-induced endocytosis of EGF receptors in intact cells.

Adaptor Protein Complex 2↗

PubMed2001

Cholecystokinin activation of 70-kDa S6 kinase in exocrine pancreas.

Cholecystokinin (CCK), a known mitogen for the exocrine pancreas, is shown to activate 70-kDa S6 kinase in isolated pancreatic acini. In this study, we examined the kinetics and cellular mechanisms of CCK-induced p70 S6 kinase activation in vivo and in vitro. Fasted mice were intraperitoneally injected with 0.01-10 microg/kg CCK analoge cerulein. Cerulein caused a concentration-dependent activation of p70 S6 kinase, with the maximal effect at 1-10 microg/kg. After 1 microg/kg cerulein administration, the kinase activity was increased at 5 min, peaked at 10 min, and subsequently decreased. Cerulein also caused a rapid and transient activation of Src. Prior administration of the tyrosine kinase inhibitor herbimycin A compeletely inhibited cerulein-induced Src activation, while the inhibition of p70 S6 kinase activity was partial. Similar results were obtained with pancreatic acinar cell line AR42J cells. These results suggest that tyrosine kinases, including Src as a possible candidate, are partly implicated in the signaling pathway of CCK-induced p70 S6 kinase activation in the exocrine pancreas.

Animals↗

PubMed2001

Differential control of cellular gene expression by diffusible and non-diffusible EGF.

Cell gene expression is affected by both the kind and mode of growth factor stimulation (diffusive vs. non-diffusive). Epidermal growth factor (EGF) was pattern-immobilized on a polystyrene plate. Although the growth of the rat phaeochromocytoma cell line PC12 is stimulated by diffusible EGF, and differentiation is stimulated by diffusible nerve growth factor (NGF), immobilized (non-diffusible) EGF stimulated PC12 differentiation. The immobilized EGF caused a long-lasting stimulation of the intracellular signal protein mitogen-associated protein MAP kinase (MAPK, also known as ERK) and p38 (a subfamily of the MAPK superfamily) in cells, as did diffusible NGF. The switching between growth stimulation and differentiation is considered to be due to the duration of the stimulus. The function of the biosignal conjugate was regulated using conjugation methodology.

Animals↗

PubMed2000

Polymorphism of cystic fibrosis gene in Japanese patients with chronic pancreatitis.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and the 5T genotype of the polythymidine tract at the exon 9 splice branch/acceptor site are shown to be associated with chronic pancreatitis in Caucasian patients. In contrast to Western countries, cystic fibrosis is extremely rare in Japan. In this study, we investigated the association of mutations or polymorphisms of the CFTR gene with chronic pancreatitis in Japanese patients. Forty-seven patients with chronic pancreatitis (alcohol-related in 31, idiopathic in 14, and familial in 2) were examined for the deltaF508 and R117H mutations and polymorphisms of intron 8. DNA was extracted from leukocytes. Mutations and polymorphisms were examined by the allele-specific polymerase chain reactions and confirmed by direct sequencing. None of the patients had deltaF508 or R117H mutations in the CFTR gene. All of 47 healthy Japanese showed the homozygous 7T/7T genotype, whereas the frequencies of 5T, 7T, and 9T alleles were 0.043, 0.894, and 0.064 in the patients, respectively. The difference in allele frequency is statistically significant. Therefore, the present study indicates the association of polymorphism of the polythymidine tract in intron 8 of the CFTR gene with chronic pancreatitis in Japanese patients.

Adult↗

PubMed2000

Alcohol and aldehyde dehydrogenase polymorphisms in Japanese patients with alcohol-induced chronic pancreatitis.

In order to clarify the genetic factors in alcohol-related chronic pancreatitis among Japanese, we determined the genotype of two major alcohol-metabolizing enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). The restriction fragment-length polymorphisms of the ADH2 and the ALDH2 genes were analyzed in 47 normal subjects and 31 patients with alcoholic pancreatitis. No significant difference between the patient and control groups was found in the ADH2 genotypes. A significant genetic difference between the two groups was found in the ALDH2 locus. The frequency of the ALDH2*1 allele was found to be 0.681 and that of the ALDH2*2 allele was 0.319 in the controls, while these values were 0.935 and 0.065 in the patients, respectively. Most of the patients (27 of 31) were ALDH2*1/2*1, only four were ALDH2*1/2*2, and none of the patients were ALDH2*2/2*2. These results indicate that genetic polymorphism of the ALDH2 gene influences the risk of developing alcoholic pancreatitis in Japanese.

Adult↗

PubMed1999

A case of acute pancreatitis complicating Salmonella enteritis.

We report a case of acute pancreatitis complicating Salmonella enteritis. A 43-yr-old woman who was admitted to our department because of Salmonella enteritis developed clinical acute pancreatitis with laboratory and radiographic signs on the fourth hospital day. She was free from symptoms on the eighth hospital day, but her elevated serum amylase and lipase levels persisted for more than 2 m.o. In this case, clinical acute pancreatitis was a complication of bacterial enteritis caused by Salmonella enteritidis, and it was characterized by onset a few days after the onset of enteritis and by sustained elevation of serum pancreatic enzyme levels.

Acute Disease↗

PubMed1998

Liposome immunoassay by long-lived fluorescence detection.

Immunoassay by fluorescence energy transfer from a europium chelate in liposome to allophycocyanin (APC) was demonstrated. Streptavidin or antibody to biotin was bonded to the liposome containing the europium chelate of 2-naphthoyltrifluoroacetone in the bilayer. When the biotin bonded to APC (APC-BT) was added to the prepared liposomes and the long-lived fluorescence (lambda ex 336 nm, lambda em 665 nm, delay time 0.05 ms, gate time 3.9 ms) was measured by a flow system, the fluorescence energy of the europium chelate was found to be transferred to APC-BT and the long-lived fluorescence intensity to increase linearly as the concentration of APC-BT (1-10 micrograms ml-1) increased. Further, the intensity decreased competitively with the concentration of biotin (0.1-100 microM) when biotin was added to the liposome solution containing a constant concentration of APC-BT.

Animals↗

PubMed1998

Supramaximal CCK and CCh concentrations abolish VIP potentiation by inhibiting adenylyl cyclase activity.

Exocrine pancreatic secretion stimulated by vasoactive intestinal polypeptide (VIP), which acts through the adenylyl cyclase-cAMP pathway, is potentiated by stimulation with other secretagogues such as CCK and carbachol (CCh). However, the potentiating effect is abolished by the same secretagogues at supramaximal concentrations. In the present study, we examined the mechanisms by which supramaximal concentrations of CCK octapeptide (CCK-8) or CCh reduce the VIP-induced potentiation of amylase secretion from isolated rat pancreatic acini. VIP-stimulated amylase secretion was potentiated by submaximal stimulatory concentrations of CCK-8 and CCh but was reduced by the same reagents at higher concentrations. Supramaximal concentrations of CCK-8 or CCh also reduced forskolin-induced potentiation of amylase release but did not reduce that induced by 8-bromo-cAMP. Moreover, supramaximal concentrations of CCK-8 or CCh inhibited VIP-stimulated intracellular cAMP production as well as adenylyl cyclase activity. 12-O-tetradecanoylphorbol 13-acetate (TPA) also reduced the magnitude of the potentiation of amylase release caused by VIP plus CCK-8 or CCh, although TPA itself decreased neither VIP-stimulated adenylyl cyclase activity nor intracellular cAMP accumulation. These results indicate that supramaximal concentrations of CCK-8 and CCh reduce the potentiating effect of VIP and forskolin on amylase secretion by inhibiting the adenylyl cyclase activity. In addition, protein kinase C is suggested to be partly implicated in this inhibitory mechanism. The mechanisms that lead to such inhibition may be interlinked but distinct from each other.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1998

Shc phosphotyrosine-binding domain dominantly interacts with epidermal growth factor receptors and mediates Ras activation in intact cells.

The adaptor protein Shc contains a phosphotyrosine binding (PTB) domain and a Src homology 2 (SH2) domain, both of which are known to interact with phosphorylated tyrosines. We have shown previously that tyrosine 1148 of the activated epidermal growth factor (EGF) receptor is a major binding site for Shc while tyrosine 1173 is a secondary binding site in intact cells. In the present study, we investigated the interaction between the PTB and SH2 domains of Shc and the activated human EGF receptor. Mutant 52-kDa Shc with an arginine-to-lysine substitution at residue 175 in the PTB domain (Shc R175K) or 397 in the SH2 domain (Shc R397K) was coexpressed in Chinese hamster ovary cells overexpressing the wild-type or mutant EGF receptors that retained only one of the autophosphorylation sites at tyrosine 1148 (QM1148) or 1173 (QM1173). Shc R397K was coprecipitated with the QM1148 and QM1173 receptors, was tyrosine-phosphorylated, and associated with Grb2 and Sos. In contrast, coprecipitation of Shc R175K with the mutant receptors was barely detectable. In cells expressing the QM1173 receptor, Shc R175K was tyrosine-phosphorylated and associated with Grb2, while association of Sos was barely detectable. In cells expressing the QM1148 receptor, tyrosine phosphorylation of Shc R175K was markedly reduced. When both Shc R175K and 46-kDa Shc R397K were coexpressed with the mutant receptors, p46 Shc R397K was dominantly tyrosine-phosphorylated. In cells expressing the wild-type receptor, Shc R397K, but not Shc R175K, translocated to the membrane in an EGF-dependent manner. In addition, Ras activity stimulated by the immunoprecipitates of Shc R397K was significantly higher than that by the immunoprecipitates of Shc R175K. The present results indicate that tyrosine 1148 of the activated EGF receptor mainly interacts with the Shc PTB domain in intact cells. Tyrosine 1173 interacts with both the PTB and SH2 domains, although the interaction with the PTB domain is dominant. In addition, Shc bound to the activated EGF receptor via the PTB domain dominantly interacts with Grb2-Sos complex and plays a major role in the Ras-signaling pathway.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Epidermal growth factor stimulates the tyrosine phosphorylation of SHPS-1 and association of SHPS-1 with SHP-2, a SH2 domain-containing protein tyrosine phosphatase.

SHPS-1 is a 120 kDa glycosylated receptor-like protein that contains immunoglobulin-like domains in its extracellular region and four potential tyrosine phosphorylation for SH2 domain binding sites in its cytoplasmic region. Epidermal growth factor (EGF) stimulated the rapid tyrosine phosphorylation of SHPS-1 and subsequent association of SHPS-1 with SHP-2, a protein tyrosine phosphatase containing SH2 domains, in Chinese hamster ovary cells overexpressing human EGF receptors. In the cells overexpressing SHPS-1, the tyrosine phosphorylation of SHPS-1 was more evident than that observed in parent cells. However, overexpression of SHPS-1 alone did not affect the activation of MAP kinase in response to EGF. These results suggest that SHPS-1 may be involved in the recruitment of SHP-2 from the cytosol to the plasma membrane in response to EGF.

Animals↗

PubMed1997

Enhancement of the mitogenic effect by artificial juxtacrine stimulation using immobilized EGF.

Mouse epidermal growth factor (EGF) was covalently conjugated with the water-soluble polymer, poly(acrylic acid) (EGF-PAA), or with the water-insoluble polymer, surface-hydrolyzed poly(methyl methacrylate) (EGF-PMMA). Immobilized EGF (EGF-PMMA) stimulated DNA synthesis in Chinese hamster ovary cells overexpressing EGF receptors in amounts that were 5 to 10% of those of free EGF required for comparable effects. In addition, the maximal mitogenic effect of EGF-PMMA was greater than that of unconjugated EGF or EGF-PAA. EGF, EGF-PAA, and EGF-PMMA induced the autophosphorylation of EGF receptors and the stimulation of mitogen-activated protein kinase. However, whereas the onset of these effects was delayed with EGF-PMMA, they persisted for much longer than those of EGF and EGF-PAA. Unlike EGF and EGF-PAA, EGF-PMMA was not associated with cells after their removal from culture and did not induce receptor internalization. Culturing cells with PMMA-immobilized EGF thus represents a model system for studying "juxtacrine" stimulation of cells by membrane-bound growth factors.

Acrylic Resins↗

PubMed1996

Interaction of Shc with adaptor protein adaptins.

The role of Shc as a substrate of receptors for growth factors and cytokines is well established. To gain further insight into the function of Shc in signal transduction, we used an affinity method to identify potential Shc-binding proteins. Incubation of bovine brain lysates with a glutathione S-transferase (GST)-Shc fusion protein immobilized on glutathione-Sepharose beads resulted in the binding of cellular proteins of approximately 115, 110, and 100 kDa as well as those of 50 and 17 kDa. Amino acid sequencing of tryptic peptides revealed that the 100-kDa protein was almost identical to beta-adaptin and that the 110- and 115-kDa proteins were almost identical to alphaA-adaptin. Using immunoblot analysis, anti-alpha-adaptin antibody recognized several proteins of 100 approximately 115 kDa, and anti-beta-adaptin antibody recognized a 100-kDa protein, suggesting that alphaA-, alphaC-, and beta-adaptins are bound to the GST-Shc fusion protein. Immunoblot analysis with anti-alpha-adaptin antibody revealed that alpha-adaptin was coimmunoprecipitated with Shc from PC12, KB, and COS cell lysates, suggesting a specific interaction of Shc and adaptins in intact cells. A binding study using mutant GST-Shc fusion proteins revealed that the collagen homologous region (amino acids 233-377) of Shc was required for adaptin binding. Conversely, the collagen homologous region of Shc inhibited the binding of adaptins to GST-Shc. In addition, adaptin was able to bind mutant fusion proteins containing amino acids 233-369, 233-355, 346-369, and 346-355 of Shc, but failed to bind a mutant containing amino acids 233-345, suggesting that amino acids 346-355 (RDLFDMKPFE) in the collagen homologous region of Shc are required for adaptin binding. Thus, this study indicates the specific interaction of Shc with alpha- and beta-adaptin components of plasma membrane adaptor proteins that are thought to be involved in receptor endocytosis.

Adaptor Protein Complex 2↗

PubMed1996

Tryptophan modulates exocrine secretory function in rat pancreatic acini.

We investigated the effect of tryptophan (Trp) on exocrine secretory function, using isolated rat pancreatic acini. Trp inhibited cholecystokinin-octapeptide (CCK-8)-stimulated amylase secretion, causing a downward shift in the dose-response curve. The inhibitory effect of Trp was dose-dependent and was observed only on the sustained secretion, there being no effect on the initial phase of amylase secretion. Trp (10mM) also inhibited amylase secretion in response to carbachol and bombesin, as well as fluoride, a potent activator of guanine-nucleotide binding proteins. Since Ca2+ influx is necessary for sustained secretion, we examined the effect of Trp on Ca2+ influx and efflux. Trp increased the CCK-8-stimulated Ca2+ influx rate without affecting Ca2+ efflux, suggesting that Trp elevates intracellular Ca2+ levels. Increasing intracellular Ca2+ levels with A23187 resulted in the inhibition of CCK-8-stimulated amylase secretion. These results indicate that Trp inhibits CCK-stimulated sustained amylase secretion, in part by increasing Ca2+ influx into acinar cells.

Amylases↗

PubMed1996

Oral glucose ingestion stimulates cholecystokinin release in normal subjects and patients with non-insulin-dependent diabetes mellitus.

The role of glucose in the regulation of plasma cholecystokinin (CCK) level was investigated in healthy control subjects and patients with non-insulin-dependent diabetes mellitus (NIDDM). Plasma CCK concentration was determined by a specific and sensitive bioassay and by a highly sensitive and reliable double-antibody radioimmunoassay using OAL-656 as an antiserum. In control subjects, ingestion of Trelan G-75 (1,200 mOsm/L,225 mL), which is equivalent to 75 g glucose as metabolic products, caused a rapid and significant increase in plasma CCK bioactivity from 1.3 +/- 0.2 to a peak of 5.8 +/- 0.6 pmol/L and immunoreactive CCK concentration from 1.2 +/- 0.1 to 4.6 +/- 0.6 pmol/L. Ingestion of 75 g glucose in 225 mL water (33.3% solution) increased plasma CCK bioactivity to a similar degree to that observed following Trelan G-75 (peak response, 4.5 +/- 0.4 pmol/L). The same volume of 0.9% NaCl solution or water failed to increase plasma CCK concentration. A smaller dose of glucose (50 b/150 mL water) increased plasma CCK concentration, although the peak level (3.0 +/- 0.5 pmol/L) was less than that observed following 75 g glucose. In patients with NIDDM, Trelan G-75 ingestion increased CCK concentration, but the peak level was lower, albeit insignificantly, than that of normal subjects. When the maximal increment of plasma CCK above the basal value was compared between control and NIDDM subjects, the differences were statistically significant (NIDDM, 3.6 +/- 0.1 pmol/L; control, 5.0 +/- 0.4; P < .01). However, integrated CCK responses to Trelan G-75 in NIDDM (165.8 +/- 15.5 pmol/120 min) were not significantly different from those in control subjects (189.8 +/- 15.9 pmol/120 min). Peak CCK bioactivity occurred within 10 to 30 minutes of ingestion, preceding the increase in glucose and insulin. These results suggest a possible effect of CCK on insulin release in humans, and that the CCK secretory response to glucose in well-controlled diabetic patients is not significantly altered.

Administration, Oral↗

PubMed1996

Ethanol inhibits CCK-induced enzyme secretion by affecting calcium-pump activity in isolated rat pancreatic acini.

The aim of this study was to clarify the effect of ethanol on stimulus-secretion coupling in pancreatic exocrine secretion. We investigated the effects of 600 mM ethanol on cholecystokinin octapeptide (CCK-8)-stimulated amylase release, cytosolic free Ca2+ concentration ([Ca2+]i) and Ca2+ fluxes using in vitro isolated rat pancreatic acini. Ethanol, given alone, stimulated both the initial and the sustained phases of amylase release. On the other hand, ethanol inhibited only the sustained phase of amylase release stimulated by CCK-8. Ethanol also inhibited amylase release in response to fluoride, a direct activator of guanine nucleotide-binding protein, suggesting that ethanol affects intracellular signal transduction molecules. Ethanol had no influences on the initial rise but increased the sustained rise in [Ca2+]i stimulated by CCK-8 and inhibited CCK-8-stimulated Ca2+ outflux without affecting Ca2+ influx. 8-Bromoguanosine 3':5'-cyclic monophosphate, a membrane-permeable analogue of cGMP regulating membrane Ca(2+)-pump activity in various cells, completely reversed the ethanol-induced inhibition of amylase release and Ca2+ outflux in response to CCK-8 as well as fluoride. Given that Ca2+ plays a critical role in stimulus-secretion coupling in pancreatic exocrine secretion, our results indicate that 600 mM ethanol inhibits CCK-8-stimulated amylase release by inhibiting Ca(2+)-pump activity on the plasma membrane.

Amylases↗

PubMed1995

EGF-induced activation of 70-kDa S6 kinase in CHO cells expressing human EGF receptors.

We investigated epidermal growth factor (EGF)-induced activation of 85-kDa/110-kDa phosphatidylinositol (PI)-3-kinase and 70-kDa S6 kinase in Chinese hamster ovary cells expressing the human EGF receptor. EGF-induced activation of p70 S6 kinase was comparable to that induced by insulin, whereas that of PI-3-kinase in anti-phosphotyrosine immunoprecipitates was very small compared with insulin. Wortmannin, a p85/p110 PI-3-kinase inhibitor, inhibited EGF-induced activation of p70 S6 kinase in a dose-dependent manner. Given that several proteins homologous to catalytic subunit of p85/p110 PI-3-kinase have been identified and that wortmannin inhibits distinct form of PI-3-kinase, the present results suggest that wortmannin-sensitive kinases that resemble catalytic subunit of p85/p110 PI-3-kinase may participate in the signaling pathway from EGF receptors to p70 S6 kinase.

Androstadienes↗

PubMed1995

Chronic oral administration of synthetic trypsin inhibitor camostate reduces amylase release from isolated rat pancreatic acini.

In the present study, we examined stimulus-secretion coupling in pancreatic acini prepared from rats given synthetic protease inhibitor camostate at a dose of 200 mg/kg body wt by an orogastric tube once a day for 10 d. Camostate treatment significantly increased pancreatic weight, protein, DNA, and enzyme contents. In acini prepared from the camostate-treated rats, responsiveness to both CCK-8 and carbamylcholine was greatly decreased with no shift in the dose-response curves compared to control acini prepared from saline-treated rats. There were no major changes in the affinity for both high- and low-affinity sites of CCK receptors, but there was a significant reduction in the capacity of low-affinity site based on acinar protein. Responsiveness to secretin in the camostate-treated rat acini was also significantly reduced compared with that in the controls. However, amylase release from the camostate-treated rat acini in response to an increase in intracellular calcium levels induced by the calcium ionophores A23187 or to an increase in intracellular cyclic 3',5'-monophosphate (cyclic AMP) levels caused by 8 bromo cyclic AMP was not significantly different from the control rat acini, suggesting that both Ca(2+)-dependent tyrosine kinase and nucleotide-activated kinases are not impaired. On the other hand, the responsiveness to phorbol ester TPA, which stimulates amylase secretion via a calcium-independent cascade by activating protein kinase C directly, was reduced in the camostate-treated rat acini compared with the controls. These results suggest the possibilities that the reduced amylase secretion in the camostate-treated rats is owing to alterations in both the transmembrane signal transduction and the phosphorylation of regulatory proteins by the Ca(2+)-independent, protein kinase C-dependent mechanisms.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1994

Grb2/Ash binds directly to tyrosines 1068 and 1086 and indirectly to tyrosine 1148 of activated human epidermal growth factor receptors in intact cells.

The activation of receptor tyrosine kinases generates tyrosine-phosphorylated recognition motifs for the binding of signaling proteins containing Src homology 2 domains. We determined the binding sites of Grb2/Ash, an Src homology 2 domain-containing adaptor protein, within epidermal growth factor (EGF) receptors, using Chinese hamster ovary cells overexpressing human EGF receptor mutants in which one of the autophosphorylation sites was retained. In intact cells, the amount of Grb2/Ash coimmunoprecipitated with mutant receptors retaining tyrosines 992, 1068, 1086, 1148, or 1173 was approximately 10, 85, 55, 50, or 20% of wild-type levels, respectively. The association of Grb2/Ash with in vitro autophosphorylated EGF receptor mutants was detectable in those retaining either tyrosines 1068 or 1086 but not in other mutants including those retaining tyrosine 1148. In peptide inhibition assay, phosphorylated peptides representing tyrosines 1068 and 1086 inhibited the binding of Grb2/Ash to in vitro autophosphorylated wild-type EGF receptors, whereas the other peptides representing tyrosines 992, 1148, and 1173 failed to inhibit the binding. Given that tyrosine 1148 of the activated EGF receptor is a major binding site of Shc (Okabayashi, Y., Kido, Y., Okutani, T., Sugimoto, Y., Sakaguchi, K., and Kasuga, M. (1994) J. Biol. Chem. 269, 18674-18678), these results indicate that tyrosines 1068 and 1086 of activated human EGF receptors are direct high affinity binding sites of Grb2/Ash and that tyrosine 1148 is an indirect binding site through Shc in intact cells.

Adaptor Proteins, Signal Transducing↗