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Biomedical subjects

G Carpenter

Publications and source records attributed to G Carpenter.

At least 73 records · Page 4Linked to original sources
PubMed1994

Potent SHC tyrosine phosphorylation by epidermal growth factor at low receptor density or in the absence of receptor autophosphorylation sites.

The importance of epidermal growth factor (EGF) receptor expression level and autophosphorylation sites in src homology and collagen protein (SHC) tyrosine phosphorylation has been studied. In contrast to EGF-induced tyrosine phosphorylation of the GTPase-activating protein for ras (rasGAP) and phospholipase C-gamma 1 (PLC-gamma 1), SHC tyrosine phosphorylation occurs at a very low receptor density in parental NIH3T3 mouse fibroblasts expressing less than 1 x 10(4) EGF receptors per cell. In transfected NIH3T3 cells expressing human EGF receptors (approximately 4 x 10(5) receptors per cell), maximal levels of SHC and PLC-gamma 1 tyrosine phosphorylation occur when approximately 4 x 10(4) receptors or more are occupied by ligand. At lower levels of receptor occupancy only SHC phosphorylation was significant. Also, EGF treatment of mouse keratinocytes, which represent a physiological target of EGF, express a low number of EGF receptors (approximately 2 x 10(4) receptors per cell), and stringently require EGF to grow, results in intense SHC tyrosine phosphorylation, compared to rasGAP or PLC-gamma 1. SHC is also efficiently tyrosine phosphorylated by an EGF receptor deletion mutant (Dc214) that is devoid of autophosphorylation sites, but which remains mitogenically responsive to EGF. The EGF receptor mutant Dc214 is able to activate the ras guanine nucleotide exchanger and phosphorylate mitogen-activated protein kinase (MAPK), presumable as a result of complex formation between tyrosine phosphorylated SHC and GRB2. These results indicate that potent EGF-induced SHC tyrosine phosphorylation can be triggered in cells having relatively few receptors. Also, our data show that EGF receptors are able to phosphorylate SHC, activate the exchange of guanine nucleotide on ras and phosphorylate MAPK by a mechanism that does not require receptor autophosphorylation sites and, therefore, the src homology 2 (SH2):phosphotyrosine-dependent interaction of SHC or GRB2 with the EGF receptor.

3T3 Cells↗
PubMed1994

Epidermal growth factor increases protein and messenger RNA expression levels of Ras GTPase activating protein.

The Ras GTPase activating protein (RasGAP) is a key regulatory enzyme in the Ras signaling pathway, which is crucial for growth factor-induced mitogenesis. In this study, it is shown that epidermal growth factor (EGF) increases RasGAP protein expression by 100-150% in NIH 3T3 cells, which overexpress the human EGF receptor, and mouse keratinocytes but does not increase RasGAP protein expression in A431 cells, where EGF does not have a mitogenic effect. In contrast, EGF does not affect the expression of other signal transduction SH2-containing proteins, such as phospholipase C gamma 1 and the p85 subunit of phosphatidylinositide 3-kinase. The growth factor-induced increase of RasGAP protein parallels an increase in RasGAP activity. EGF stimulates RasGAP protein synthesis and does not affect its degradation rate. The mechanism for RasGAP protein induction by EGF involves an increase in rasGAP mRNA levels. The growth factor-stimulated up-regulation of rasGAP is a delayed response, since the increase in the mRNA levels and protein synthesis rate begin after 3 h and reach maximal values between 9 and 24 h of growth factor treatment. EGF fails to increase rasGAP mRNA levels in the presence of cycloheximide, suggesting that this effect is dependent on de novo protein synthesis. However, cycloheximide alone is able to increase by 6-fold rasGAP mRNA expression. Since EGF does not modify rasGAP mRNA stability, the increase in rasGAP mRNA expression is likely due to an increase in transcription of the rasGAP gene.

Animals↗
PubMed1993

Tyrosine phosphorylation of ras GTPase-activating protein does not require association with the epidermal growth factor receptor.

The importance of the carboxyl-terminal domain of the epidermal growth factor (EGF) receptor and its five autophosphorylation sites in the in vivo interaction and tyrosine phosphorylation of the ras GTPase-activating protein (rasGAP) has been investigated, using NIH 3T3 cells transfected with mutant EGF receptors. Phosphorylation of rasGAP by EGF receptor mutants, in which one to four autophosphorylation sites (Tyr-1173, -1148, -1086, and -1068) were mutated to phenylalanine, was reduced by 50-60% compared to the wild-type receptor. Elimination of these four autophosphorylation sites by truncation of 123 carboxyl-terminal residues of the EGF receptor paralleled results obtained with point mutants. Substantial inhibition (about 90%) of rasGAP tyrosine phosphorylation by the EGF receptor occurred only when the remaining autophosphorylation site (Tyr-992) was mutated, in the context of this truncated receptor or in the full-length receptor mutated at all four other autophosphorylation sites. However, a point mutation of only Tyr-992 in the full-length receptor suppressed tyrosine phosphorylation of rasGAP only by 50%. In contrast, an EGF receptor lacking the last 214 amino acid residues (Dc214), which encompasses all five autophosphorylation sites, phosphorylated rasGAP to the same extent as the wild-type receptor. However, this truncated receptor was significantly impaired in its capacity to phosphorylate phospholipase C-gamma 1. Interestingly, while EGF receptor autophosphorylation sites are required for EGF-induced rasGAP association with the receptor, maximal phosphorylation of rasGAP by the truncated receptor Dc214 occurred without detectable formation of receptor-rasGAP complexes. Furthermore, the capacity of mutated EGF receptors to bring about focal transformation was correlated with their capacity to phosphorylate rasGAP.

3T3 Cells↗
PubMed1993

The regulation of phospholipase C-gamma 1 by phosphatidic acid. Assessment of kinetic parameters.

A survey of lipids revealed that the anionic phospholipid phosphatidic acid activates both control and tyrosine-phosphorylated PLC-gamma 1. The mechanism by which phosphatidic acid activates both forms of PLC-gamma 1 was investigated using kinetic analysis. In the presence of phosphatidic acid, the substrate concentration response for control PLC-gamma 1 changes from sigmoidal to hyperbolic, while the cooperativity index decreases from 2.5 for control to 1.0 for tyrosine-phosphorylated PLC-gamma 1. The primary influence of phosphatidic acid on the control enzyme is on the cooperativity index and not the association of PLC-gamma 1 with substrate micelles, as phosphatidic acid had little effect on the micellar association constant, Ks. Phosphatidic acid also increases the activity of the tyrosine phosphorylated form of the enzyme. This increase is reflected in a decrease in the Km from 0.3- to 0.03-mol fraction phosphatidylinositol 4,5-bisphosphate. Phosphatidic acid has no effect on the Ks of the tyrosine-phosphorylated enzyme. From this data it is concluded that phosphatidic acid appears to activate PLC-gamma 1 by acting as an allosteric modifier.

Allosteric Regulation↗
PubMed1993

Epidermal growth factor stimulates substrate-selective protein-tyrosine-phosphatase activity.

This study investigates the regulation of protein-tyrosine-phosphatase (PTPase; EC 3.1.3.48) activity by epidermal growth factor (EGF). Cytosol from EGF-treated A-431 human epidermoid carcinoma cells was used as a source of PTPase activity, and tyrosine-phosphorylated ErbB2, EGF receptor, phospholipase C-gamma 1, and the Ras GTPase-activating protein were used as substrates to monitor PTPase activity. EGF stimulated PTPase activity that was selective toward these substrates, as it dephosphorylated ErbB2 and the EGF receptor, but not phospholipase C-gamma 1 and the Ras GTPase-activating protein. EGF stimulated PTPase activity in several cell lines, regardless of EGF receptor number, and the activity was localized in the cytosol. The dephosphorylation activity in vitro was dependent on the presence of reducing agents and was inhibited by orthovanadate. Agonists such as phorbol 12-myristate 13-acetate, isoproterenol, or ATP were unable to stimulate PTPase activity. Physiological relevance is indicated by experiments showing that EGF treatment of a human mammary cancer cell line rapidly induced the dephosphorylation of ErbB2.

3T3 Cells↗
PubMed1993

Interaction of activated EGF receptors with coated pit adaptins.

The epidermal growth factor (EGF) receptor interacts with plasma membrane-associated adapter proteins during endocytosis through coated pits. Almost 50 percent of the total pool of alpha-adaptins was coimmunoprecipitated with the EGF receptor when A-431 cells were treated with EGF at 37 degrees C, but not at 4 degrees C. Partial proteolysis of alpha-adaptin suggested that the amino-terminal domain is the region that associates with the EGF receptor. The extent of receptor-adaptin association was increased in cells depleted of potassium to block endocytosis. These data suggest that receptor-adaptin association occurs in intact cells before coated pits are fully assembled.

Adaptor Protein Complex alpha Subunits↗
PubMed1993

Non-catalytic activation of phospholipase C-gamma 1 in vitro by epidermal growth factor receptor.

To investigate the possible functional role of epidermal growth factor (EGF) receptor-phospholipase C-gamma 1 (PLC-gamma 1) complexes, we have measured PLC-gamma 1 activity in vitro in the absence or presence of purified EGF receptor. Immunoprecipitates of PLC-gamma 1 from control A-431 cells were incubated without or with purified EGF receptor in the absence or presence of ATP. Under these conditions the EGF receptor increased non-tyrosine-phosphorylated PLC-gamma 1 activity 3-4-fold in the absence or presence of ATP, but increased tyrosine-phosphorylated and activated PLC-gamma 1 by only 20-50%. Both basal and autophosphorylated forms of the purified EGF receptor increased the activity of the non-tyrosine-phosphorylated PLC-gamma 1, and stoichiometric levels of purified receptor were required to increase PLC activity. Other tyrosine kinases such as the platelet-derived growth factor receptor and erbB-2, but not the insulin receptor, also stimulated PLC-gamma 1 activity. PLC-gamma 1 activity could be activated with the kinase-negative EGF receptor, but a C-terminal truncated receptor was much less effective. Purified EGF receptor could also activate PLC-beta 1, but with a much decreased potency compared with PLC-gamma 1. Our results suggest that in vitro the EGF receptor can increase PLC-gamma 1 activity independently of tyrosine phosphorylation.

3T3 Cells↗
PubMed1993

Differential heat stress stability of epidermal growth factor receptor and erbB-2 receptor tyrosine kinase activities.

The epidermal growth factor (EGF) and erbB-2 receptors are structurally related membrane-bound tyrosine kinases. While these proteins exhibit close sequence homology, 50% overall and 80% in the tyrosine kinase domains, they respond very differently to heat stress. In NIH-3T3 or NR6 cells transfected with wild-type EGF-R and incubated at 37 degrees C or heat shocked at 46 degrees C, EGF binds to its receptor and stimulates receptor autophosphorylation to equivalent extents. At 46 degrees C, however, the basal tyrosine kinase activity of the wild-type erbB-2 receptor is rapidly lost. When cells containing chimeric receptors composed of the EGF-R extracellular domain and intracellular domain of erbB-2 were heat stressed, 125I-EGF bound to the receptors, but did not stimulate receptor autophosphorylation. The decline in EGF-stimulated chimeric erbB-2 receptor autophosphorylation is dependent on the length of heat shock, with nearly 100% of the kinase activity lost after 60 min at 46 degrees C. The loss of chimeric receptor erbB-2 kinase activity is not due to degradation of receptor protein, nor is it attributable to a specific transmembrane domain from either the EGF or erbB-2 receptors. Sensitivity of erbB-2 to heat stress is also not a result of denaturation of this receptor's carboxy-terminal domain. Insertion of the erbB-2 tyrosine kinase domain into the EGF-R confers heat stress sensitivity to the resultant chimeric receptor. Thus, although the EGF-R and erbB-2 kinase domains show a high degree of homology, the secondary/tertiary structures of these domains would seem to be stabilized in distinct manners.

3T3 Cells↗
PubMed1993

EGF: new tricks for an old growth factor.

During the past year, the biology of epidermal growth factor (EGF) has been investigated in lower organisms (Caenorhabditis elegans, Drosophila and bacteria). These experiments have produced some surprising results: the identification of defects produced by mutation of EGF-like genes; the role of EGF receptors in bacterial invasion; and the role of EGF-like precursors as receptors for a bacteria toxin.

Animals↗
PubMed1993

The prognostic utility of delayed-type hypersensitivity skin testing in the evaluation of HIV-infected patients. Military Medical Consortium for Applied Retroviral Research.

Many reagents and techniques have been used for delayed-type hypersensitivity (DTH) skin testing in the evaluation of HIV-infected patients, resulting in varied interpretation of the utility of DTH skin testing in this population. We report the development of a simple algorithm for selection of DTH antigens and the clinical relevance of DTH skin testing in HIV disease. Antigens and concentrations for testing were first evaluated in a demographically matched, HIV-negative, immunologically healthy population. The testing scheme was then applied to the HIV population of interest for 5 years at several clinical sites. The antigens and concentrations selected resulted in 100% reactivity to two or more antigens in the HIV-negative cohort. Anergy is thus a distinct immunologic abnormality. Although some correlation (r2 = 0.6) of skin test reactivity and CD4 cell count was found in a cohort of HIV-infected individuals, anergy was found to be independently predictive of the development of symptomatic late-stage disease (Walter Reed Stage 6), AIDS, or death. This stepwise evaluation of skin testing and reagents has led to the modification of the skin testing protocol by defining the minimum number of antigens required and establishing the independent prognostic role of DTH skin testing in the evaluation of HIV-infected patients. The addition of mumps (40 CFU/ml), tetanus (1:10), and candida (1:10) to the purified protein derivative (PPD) skin test provides the critical controls to evaluate the status of PPD skin test in HIV-infected individuals as well as to provide a useful and prognostic clinical immunology evaluation.

Adult↗
PubMed1993

The carboxyl terminus of epidermal growth factor receptor/erbB-2 chimerae is internalization impaired.

The endocytosis of gp185erbB-2 was studied using chimeric receptors in which the intracellular domain of erbB-2, or subdomins thereof, was substituted for the corresponding regions of the epidermal growth factor (EGF) receptor. Chimeric and wild-type EGF or erbB-2 receptors were expressed in mouse NIH3T3 or NR6 fibroblasts and in a human mammary adenocarcinoma cell line, MDAMB-134. The rate of EGF-induced internalization for the chimera consisting of the extracellular EGF receptor domain and intracellular erbB-2 domain was reduced three- to fourfold compared with the wild-type EGF receptor. The low rate of internalization of the chimeric receptor resulted in impaired down-regulation and degradation of the receptor. Substitution of the carboxyl terminus of erbB-2 for the corresponding region of the EGF receptor caused a similar decrease of receptor endocytosis, whereas substitution of the erbB-2 tyrosine kinase domain did not affect internalization and down-regulation. Since the tyrosine kinase of the internalization-defective chimeric receptors could be activated by EGF, kinase activity and autophosphorylation of erbB-2 do not appear to be sufficient for a maximum rapid internalization of the chimeric receptors. These results suggest that the carboxyl terminus of erbB-2 either does not possess all the signals required for the rapid internalization or contains an inhibitory signal for rapid internalization.

Amino Acid Sequence↗
PubMed1992

Activity of the epidermal-growth-factor receptor and phospholipase C-gamma 1 in heat-stressed fibroblasts and A-431 cells.

A variety of changes in the functions of specific plasma-membrane components have been reported in cells exposed to a heat shock. In this study, we examined the consequences of heat stress on epidermal-growth-factor (EGF)-induced receptor autophosphorylation and receptor-mediated tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1), a cellular substrate. Although the tyrosine kinase activity of the EGF receptor is rapidly inactivated at 45 degrees C in vitro [Carpenter, King & Cohen (1979) J. Biol. Chem. 254, 4884-4891], EGF stimulates autophosphorylation of its receptor in both A-431 cells and human fibroblasts after a prolonged heat shock. Phosphoamino acid analysis of the receptor reveals an EGF-induced increase in phosphotyrosine and phosphoserine at 46 degrees C. EGF also stimulates the phosphorylation of phospholipase C-gamma 1 and induces the formation of inositol phosphates under heat-shock conditions. 125I-EGF binding and internalization in A-431 cells is not decreased during incubations at 46 degrees C for up to 90 min. EGF-induced dimerization of EGF receptors on the cell surface is preserved during heat shock. Though EGF-receptor-mediated endocytosis is not inhibited by elevated temperature, the degradation of internalized 125I-EGF is dramatically decreased. These results indicate that, aside from ligand degradation, the EGF-mediated pathway of signal transduction through phospholipase C-gamma 1 remains remarkably intact during conditions of extreme cellular stress.

Animals↗
PubMed1992

Elevated membrane association of phospholipase C-gamma 1 in MDA-468 mammary tumor cells.

We have examined the distribution of phospholipase C-gamma 1 (PLC-gamma 1) between membrane and cytosolic fractions in several cell lines. In MDA-468 cells, which are derived from a human breast tumor, greater than one-half of the total PLC-gamma 1 is associated with the membrane fraction of the cell. Unlike the situation in A-431 cells [G. Todderud, M. I. Wahl, S. G. Ree, and G. Carpenter, Science, 248: 296-298, 1990], epidermal growth factor (EGF) stimulation of MDA-468 cells does not result in significantly increased PLC-gamma 1 association with membranes. Immunoblot analysis reveals low levels of phosphotyrosine in PLC-gamma 1 and EGF receptors in unstimulated MDA-468 cells and greatly increased phosphotyrosine levels in these proteins as a result of EGF stimulation of the cells. We conclude that autocrine activation of EGF receptors is not responsible for the elevated association of PLC-gamma 1 with membranes in these cells.

Animals↗
PubMed1992

Growth factor stimulation of phospholipase C-gamma 1 activity. Comparative properties of control and activated enzymes.

We demonstrated previously tyrosine phosphorylation-dependent modulation of phospholipase C-gamma 1 (PLC-gamma 1) catalytic activity (Nishibe, S., Wahl, M. I., Hernandez-Sotomayor, S. M. T., Tonks, N. K., Rhee, S. G., and Carpenter, G. (1990) Science 250, 1253-1256). The increase in PLC-gamma 1 catalytic activity in A-431 cells occurs rapidly, with maximal activation 5 min after epidermal growth factor (EGF) stimulation. Certain other growth factors (fibroblast growth factor, platelet-derived growth factor) also stimulate PLC-gamma 1 catalytic activity, whereas insulin does not. A similar increase in PLC-gamma 1 specific activity (2-3-fold) was observed in both soluble (cytosol) and particulate (membrane) preparations from EGF-treated cells. Tyrosine-phosphorylated PLC-gamma 1 was detected in both cytosol and membrane fractions in lysates from EGF-treated A-431 cells, but the proportion of tyrosine-phosphorylated PLC-gamma 1 was higher in the cytosol (approximately 50%) than in the membrane (approximately 20%). Because a micellar concentration of the non-ionic detergent Triton X-100 allows detection of the tyrosine phosphorylation-dependent increase in PLC-gamma 1 catalytic activity in this assay, we evaluated the kinetic properties of PLC-gamma 1, immunoprecipitated from cytosol of control or EGF-treated cells, using substrate, phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2), solubilized in Triton X-100 at various molar ratios. The behavior of the control enzyme differed from the EGF-activated enzyme with respect to both Ks and Km. The control enzyme has a 7.5-fold higher Ks value than the activated enzyme (1.5 mM as compared with 0.22 mM). Activation by EGF is also a positive allosteric modifier of PLC-gamma 1-catalyzed PtdIns 4,5-P2 hydrolysis, i.e. the activated enzyme displayed apparent Michalis-Menton kinetics, with a Km of 0.6 mol fraction PtdIns 4,5-P2, whereas the control enzyme displayed sigmoidal kinetics with respect to PtdIns 4,5-P2 hydrolysis. At low substrate mol fractions (e.g. 0.07), the reaction velocity of the control enzyme was 4-fold lower than the activated enzyme. However, at a high substrate mol fraction (e.g. 0.33), the estimated maximal reaction velocities (Vmax) for both forms of PLC-gamma 1 were equivalent. PLC-gamma 1 activity from both control and EGF-treated cells was stimulated by increasing nanomolar Ca2+ concentrations. Although the catalytic activity of PLC-gamma 1 from EGF-treated cells was greater than control PLC-gamma 1 at every Ca2+ concentration tested, the relative stimulation of activity was markedly greater at Ca2+ concentrations above approximately 300 nM.

3T3 Cells↗
PubMed1992

Multiple autophosphorylation sites of the epidermal growth factor receptor are essential for receptor kinase activity and internalization. Contrasting significance of tyrosine 992 in the native and truncated receptors.

The role of epidermal growth factor (EGF) receptor autophosphorylation sites in the regulation of receptor functions has been studied using cells transfected with mutant EGF receptors. Simultaneous point mutation of 4 tyrosines (Y1068, Y1086, Y1148, Y1173) to phenylalanine, as well as removal of these sites by truncation of the carboxyl-terminal 123 amino acid residues, resulted in reduced receptor phosphorylation of an in vivo specific substrate phospholipase C-gamma 1 to less than 50% compared to the wild-type receptor. The internalization rate constant Ke was also significantly lower in these mutants (0.15/min) compared to cells transfected with wild-type receptor (0.27/min). Additional mutation of tyrosine 992 to phenylalanine in the truncated receptor mutant (Dc-123F) further decreased the receptor internalization rate to a minimal level (ke = 0.07-0.10/min), equivalent to the ke measured for cells expressing kinase-negative receptor (A721). Moreover, tyrosine kinase activity of the Dc-123F receptor toward phospholipase C-gamma 1, compared to wild-type receptor, was reduced by 90%. Taken together, these results show that EGF receptor lacking five autophosphorylation sites functions similar to a kinase-negative receptor. Mutation of tyrosine residue Y992 alone in the context of full length EGF receptor, however, did not affect receptor internalization or kinase activity toward phospholipase C-gamma 1. These data indicate that tyrosine 992 is critical for substrate phosphorylation and internalization only in the context of the truncated receptor, and that minor autophosphorylation sites, such as Y992, may act as compensatory regulatory sties in the absence of the major EGF receptor autophosphorylation sites.

Animals↗
PubMed1992

Analysis of the influences of the E5 transforming protein on kinetic parameters of epidermal growth factor binding and metabolism.

The E5 protein of the bovine papillomavirus induces cellular transformation when transfected into NIH 3T3 cells, and the extent of focal transformation is enhanced by cotransfection with the epidermal growth factor (EGF) receptor (Martin et al., Cell 59:21-32, 1989). To determine whether E5 affects EGF:receptor interactions we analyzed the kinetics of 125I-EGF processing using a mathematical model that enabled us to evaluate rate constants for ligand association (ka), dissociation (kd), internalization (ke), recycling (kr), and degradation (kh). These rate constants were measured in NIH 3T3 cells transfected with the human EGF receptor (ER cells) and in cells transfected with both the EGF receptor and E5 (E5/ER cells). We found that the rate constant for 125I-EGF association ka was significantly decreased in E5/ER cells, but was apparently occupancy-independent in both cell lines. The 125I-EGF dissociation rate constant kd was significantly lower in E5 transformed cells, and increased with occupancy in both cell lines. This suggests that E5 alters the receptor before or during EGF binding so that ligand association is slower; however, once complexes are formed, EGF is bound more tightly to the receptor. Rate constants for internalization ke were also found to be occupancy-dependent, although at a given level of occupancy ke was similar for both cell lines. Also, there was no apparent effect of E5 on the recycling rate constant kr. The 125I-EGF degradation rate constant kh was 30% lower in E5 transformed cells, and was occupancy-independent. The overall effect of E5 is to stabilize intact EGF:receptor complexes which may alter mitogenic signaling of the receptor.

Binding, Competitive↗
PubMed1992

Epidermal growth factor receptor: elements of intracellular communication.

While EGF has an important function in cell growth regulation, the molecular mechanisms by which intracellular signal connect the EGF: receptor complex on the plasma membrane with the initiation of DNA synthesis and mitogenesis is not well understood. The discovery that rasGAP, PI-3 kinase and PLC-gamma 1 are substrates for the EGF receptor tyrosine kinase has provided a beginning in understanding the biochemistry underlying growth factor receptor transduction.

ErbB Receptors↗