Biomedical subjects

C F Fox

Publications and source records attributed to C F Fox.

At least 37 records · Page 2Linked to original sources

PubMed1986

Differential phosphorylation of the progesterone receptor by insulin, epidermal growth factor, and platelet-derived growth factor receptor tyrosine protein kinases.

Purified preparations of insulin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) receptors were compared for their abilities to phosphorylate purified hen oviduct progesterone receptors. The specific activities of all three peptide hormone-induced receptor kinases were first defined using a synthetic tridecapeptide tyrosine protein kinase substrate. Next, equivalent ligand-activated activities of the three receptor kinases were tested for their abilities to phosphorylate hen oviduct progesterone receptor. Both the insulin and EGF receptors phosphorylated progesterone receptor at high affinity, exclusively at tyrosine residues and with maximal stoichiometries that were near unity. In contrast, the PDGF receptor did not recognize progesterone receptor as a substrate. Insulin decreased the Km of the insulin receptor for progesterone receptor subunits as substrates, but had no significant effect on Vmax values. On the other hand, EGF increased the Vmax of the EGF receptor for progesterone receptor subunits as substrates. Phosphorylation of progesterone receptor by the insulin and EGF receptor kinases differed in two additional ways. 1) EGF-activated receptor phosphorylated the 80- and 105-kDa progesterone receptor subunits to an equal extent, whereas insulin-activated receptor preferentially phosphorylated the 80-kDa subunit. 2) Phosphopeptide fingerprinting analyses revealed that while insulin and EGF receptors phosphorylated one identical major site on both progesterone receptor subunits, they differed in their specificities for other sites.

Animals↗

PubMed1984

cAMP-dependent protein kinase stimulates epidermal growth factor-dependent phosphorylation of epidermal growth factor receptors.

Epidermal growth factor (EGF)-dependent transfer of radiolabeled phosphate from [gamma-32P]ATP to 160-kDa EGF receptor solubilized from human epidermoid carcinoma A431 cell surface membranes was stimulated up to 3-fold by addition of 3',5'-cAMP and purified cAMP-dependent protein kinase. Phosphorylation of EGF receptors was stimulated to the same extent when cAMP-dependent protein kinase catalytic subunit was substituted for 3',5'-cAMP and cAMP-dependent protein kinase. Phosphoamino acid analysis revealed that the extent of phosphorylation of EGF receptor at tyrosine residues was the same regardless of whether cAMP-dependent protein kinase catalytic subunit was present in or omitted from the system. Increased EGF receptor phosphorylation occurring in response to cAMP-dependent protein kinase catalytic subunit was accounted for by phosphorylation at serine or threonine residues. In samples phosphorylated in the presence of cAMP-dependent protein kinase catalytic subunit, phosphate was present in tyrosine, serine, and threonine in a ratio of 32:60:8. Two-dimensional mapping of radiolabeled phosphopeptides produced from EGF receptors by digestion with trypsin revealed the generation of one additional major phosphoserine-containing peptide when cAMP-dependent protein kinase was present with EGF in the EGF receptor kinase system. Degradation of 160-kDa EGF receptors to a 145-kDa form by purified Ca2+-activated neutral protease produced a 145-kDa fragment with phosphoserine content increased over that present initially in the 160-kDa precursor.

Adenosine Triphosphate↗

PubMed1984

Epidermal growth factor and potent phorbol tumor promoters induce epidermal growth factor receptor phosphorylation in a similar but distinctively different manner in human epidermoid carcinoma A431 cells.

When human epidermoid carcinoma A431 cells labeled with 32Pi to steady state specific activity were treated either with epidermal growth factor (EGF) or with active phorbol ester tumor promoters such as 12-O-tetradecanoylphorbol 13-acetate (TPA) or phorbol 12,13-dibutyrate, labeling of 160 kDa EGF receptors isolated by immunoprecipitation with monoclonal anti-EGF receptor IgG was increased 2- to 3-fold. These treatments produced no significant increase in 32Pi labeling of acid-precipitable material present in detergent extracts of the cells. Phosphoamino acid analysis of radiolabeled EGF receptors isolated from these cells revealed several differences: the relative abundance of phosphotyrosine in EGF receptors was increased in cells treated with EGF, but decreased in cells treated with TPA; the overall relative abundance of phosphothreonine in EGF receptors was decreased in cells treated with EGF, but remained constant within the limits of experimental detection in cells treated with TPA. Two-dimensional mapping of the radiolabeled phosphopeptides produced from EGF receptors isolated by immunoprecipitation and treated with trypsin revealed 9 independent labeled regions, 2 of which contained phosphothreonine and were present only in EGF- or TPA-treated cells. These two phosphopeptide regions were more highly labeled in cells treated with TPA than with EGF.

Carcinogens↗

PubMed1984

Progesterone receptor subunits are high-affinity substrates for phosphorylation by epidermal growth factor receptor.

Purified preparations of epidermal growth factor (EGF) receptor were used to test hen oviduct progesterone receptor subunits as substrates for phosphorylation catalyzed by EGF receptor. Both the 80-kilodalton (kDa) (A) and the 105-kDa (B) progesterone receptor subunits were phosphorylated in a reaction that required EGF and EGF receptor. No phosphorylation of progesterone receptor subunits was observed in the absence of EGF receptor, even when Ca2+ was substituted for Mg2+ and Mn2+. Phospho amino acid analysis revealed phosphorylation at tyrosine residues, with no phosphorylation detectable at serine or threonine residues. Two-dimensional maps of phosphopeptides generated from phosphorylated 80- or 105-kDa subunits by tryptic digestion revealed similar patterns, with resolution of two major, several minor, and a number of very minor phosphopeptides. The Km of progesterone receptor for phosphorylation by EGF-activated EGF receptor was 100 nM and the Vmax was 2.5 nmol/min per mg of EGF receptor protein at 0 degrees C. The stoichiometry of phosphorylation/hormone binding for progesterone receptor subunits was 0.31 at ice-bath temperature and approximately 1.0 at 22 degrees C.

Amino Acid Sequence↗

PubMed1984

Purified plasma membranes inhibit polypeptide growth factor-induced DNA synthesis in subconfluent 3T3 cells.

Plasma membranes derived from NR-6 cells, a variant line of Swiss mouse 3T3 cells that does not have cell surface receptors for epidermal growth factor (EGF), inhibited EGF-induced stimulation of DNA synthesis by 50% in serum-starved, subconfluent 3T3 cells. Membranes derived from SV3T3 cells were much less effective in inhibiting EGF-induced DNA synthesis. This inhibition on DNA synthesis by NR-6 membranes was not a direct effect of membranes on EGF, nor could it be overcome by high concentrations of EGF. NR-6 membranes were most effective when added 3 h before EGF addition and had little effect when added 2 h or more after EGF. NR-6 membranes also reduced the stimulation of DNA synthesis induced by platelet-derived growth factor or fibroblast growth factor in serum-starved 3T3 cells. These findings indicate that membrane-membrane interactions between nontransformed cells may diminish their ability to proliferate in response to serum polypeptide growth factors.

Animals↗

PubMed1983

Calcium-mediated degradation of epidermal growth factor receptor in dislodged A431 cells and membrane preparations.

Calcium-activated neutral protease has been purified partially from A431-V1 cells, a high passage variant of human epidermoid carcinoma A431 cells having extraordinary activity of this protease. This activity cleaves Mr = 160,000 epidermal growth factor receptors, converting them to a Mr = 145,000 form in detergent-treated membrane preparations, but not in intact cells or membrane vesicles. Properties of this activity, including molecular weight, resemble those of previously described Ca2+-activated neutral proteases. A431-V1 cell Ca2+-activated neutral protease action on epidermal growth factor receptors required 0.5 mM Ca2+ for half-maximal activity and the optimal pH was 6.5. Degradation was inhibited completely by Ca2+ chelators (EDTA and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) and sulfhydryl group specific reagents (iodoacetate and 5,5'-dithiobis-(2-nitrobenzoic acid] and was inhibited partially by leupeptin. Degradation of epidermal growth factor receptors by Ca2+-activated neutral protease or trypsin was compared in intact cells, membrane vesicles, detergent-solubilized membranes, and membranes subjected to hypotonic shock in solutions containing protease. Results support the hypothesis that protease-susceptible domains of epidermal growth factor receptors, including the Ca2+-activated neutral protease-sensitive domain, are located on the cytosolic surface. The localization of Ca2+-activated neutral protease in A431-V1 cells is entirely cytosolic, making it available in cells to the protease-susceptible site on epidermal growth factor receptor substrates.

Animals↗

PubMed1983

Epidermal growth factor and epidermal growth factor receptor-dependent phosphorylation of a Mr = 34,000 protein substrate for pp60src.

A Mr = 34,000 protein present in the 100,000 X g supernatant fraction from A431 human epidermoid carcinoma cells is the major radiolabeled phosphate acceptor from [gamma-32P]ATP in a cell-free system requiring epidermal growth factor (EGF) and EGF receptor kinase. This protein is immunoprecipitated by IgG directed against avian Mr = 34,000 cellular substrate for pp60src. Phosphoamino acid analysis of the Mr = 34,000 protein labeled with 32Pi from [gamma-32P]ATP in a cell-free system requiring EGF and EGF receptor kinase yielded radiolabeled phosphotyrosine with no detectable radioactivity in phosphoserine or phosphothreonine.

Carcinoma, Squamous Cell↗

PubMed1982

Receptor remodeling and regulation in the action of epidermal growth factor.

Epidermal growth factor (EGF) initiates a wide variety of events when added to responsive cultured cells. These range from early events requiring only brief exposure to EGF, e.g., stimulation of transport of amino acids or ions, to later events such as commitment of cells to a round of DNA synthesis, a process requiring 6 h or more of continuous exposure to hormone. EGF binding is followed first by phosphorylation of EGF receptors, which can be detected in purified membranes and permeabilized cells, and then by internalization and proteolytic processing of receptors in lysosomes. Native 160,000-dalton EGF receptors contain a site that is not exposed on the cell surface and is highly sensitive to cleavage by an endogenous protease, which yields a 145,000-dalton receptor fragment that retains phosphate acceptor activity. Cleavage of receptor at a trypsin-sensitive site, also not exposed to the cell surface, yields a 115,000-dalton fragment that binds EGF, but contains no phosphorylated species. The data indicate that the phosphate acceptor sites on EGF receptors are localized on a 45,000-dalton cytosolic region.

Animals↗

PubMed1981

Neuropsychological correlations of anorexia nervosa.

Fifteen anorectic individuals were compared with a group of other psychiatric disorders with respect to the incidence of deviation in cognitive performance on a neuropsychological screening battery. A significant incidence of deviations in cognitive performance was found for the anorectic patients. The results are discussed in terms of possible neuropsychological implications that warrant future investigation.

Adult↗

PubMed1980

Modulation of epidermal growth factor receptors on 3T3 cells by platelet-derived growth factor.

Platelet-derived growth factor does not compete with epidermal growth factor (EGF) for binding to EGF receptors on the murine 3T3 cell surface, but it modulates EGF receptors in two ways: (i) it induces a transient down regulation of EGF receptors and (ii) it inhibits EGF-induced down regulation of EGF receptors. These data suggest a common cellular internalization mechanism for the receptors for both hormones.

Animals↗

PubMed1980

Direct linkage of EGF to its receptor: characterization and biological relevance.

A small portion of the 125I-EGF that binds specifically to intact cells or isolated membrane from a variety of sources becomes directly and irreversibly linked to EGF receptors. This provides a simple technique for affinity labeling the EGF receptor. Membranes isolated from the human epidermoid carcinoma cell line A431, which possesses extraordinarily high numbers of EGF receptors, gave rise to three major direct linkage complexes of MW = 160,000, 145,000, and 115,000. The time course for information of each is similar, showing that 125I-EGF can form direct linkage complexes with several preexisting forms of the EGF receptor. The direct linkage of EGF to receptor is slow in comparison to 125I-EGF binding, but both processes have similar susceptibilities to competition by unlabeled EGF. EGF was modified chemically with the amino site-specific reagent, N-hydroxysuccinimidyl biotin. The biotinyl-EGF had a reduced capacity to engage in direct linkage complex formation with no concomitant reduction in its ability to bind to EGF receptors. Since native and biotinyl EGF have identical abilities to stimulate the uptake of 3H-thymidine into DNA when incubated with cultured murine 3T3 cells, the direct linkage of EGF to its receptor does not appear to play an important role in EGF-stimulated mitogenesis.

Animals↗

PubMed1980

Controlled proteolysis of EGF receptors: evidence for transmembrane distribution of the EGF binding and phosphate acceptor sites.

A small quantity of the 125I-EGF (epidermal growth factor) bound specifically to EGF receptors on the human epidermoid carcinoma cell line A431 associates covalently. The direct linkage complex formed migrates during gel electrophoresis as a single diffuse band of MW = 160,000-170,000. In contrast, direct linkage complexes of 160,000, 145,000, and 115,000 daltons are formed when EGF is incubated with membranes isolated from these cells; these arise from EGF receptor modification during membrane isolation. None of these modifications affected the affinity of the EGF binding site for 125I-EGF. The electrophoretic mobilities of the MW = 160,000 and 145,000 direct linkage complexes were similar to those of the major 32Pi-labeled products of the EGF-stimulated phosphorylation reaction described by Carpenter et al [Nature 276:409-410, 1978], indicating that proteolytic fragments of EGF receptors are the major phosphate acceptors in this reaction. EGF receptors on intact A431 cells accepted phosphate effectively from gamma-32Pi-ATP only when the cells were permeabilized with lysolecithin. This shows that the EGF binding and phosphate acceptor sites lie on opposing faces of the membrane. When the 145,000 dalton form of receptor is labeled with EGF or 32Pi and the labeled peptides subjects to tryptic hydrolysis under identical conditions, all phosphate is lost from high molecular weight products under conditions where the EGF-receptor covalent complex is converted largely to a 115,000 dalton form. This suggests that the phosphate acceptor site lies on the cytoplasmic side of the membrane on a region of receptor extending 30,000 daltons from the 115,000 dalton fragment containing the EGF binding site.

Binding Sites↗

PubMed1980

Properties of receptors for epidermal growth factor in detergent solution: evidence for heterogeneous aggregated states.

Between 60% and 100% of epidermal growth factor (EGF) binding activity was recovered from membranes of the A431 human epidermoid carcinoma cell line treated with solutions containing the nonionic detergent Triton X-100. Approximately half of the recovered binding activity was sedimented at low centrifugal force and hence was operationally insoluble in nonionic detergent solution. Receptors in both the detergent-soluble and -insoluble fractions displayed similar affinities for 125I-EGF, and the values were in good agreement with those obtained for receptors in untreated membranes. The receptors in both fractions also formed identical direct linkage complexes with 125I-EGF in similar yield, providing no evidence for partitioning of different molecular species of EGF receptors in the detergent-soluble membrane fraction of Sepharose 6-B revealed heterogeneity of 125I-EGF binding activity; the smallest and most monodisperse peak of activity resolved by this technique was eluted at a Strokes radius of 95 A. Operationally soluble 125I-EGF binding activity also behaved heterogeneously during velocity sedimentation; more than half the activity sedimented more rapidly than the apparently monidisperse, 7S form. An average of less than half the nonionic detergent-solubilized activity recovered from 10 independent membrane preparations behaved as an apparently monodisperse entity. Since a maximum of 60% of 125I-EGF binding activity was operationally soluble, less than 25% of the total EGF binding activity was recovered in an apparently monodisperse form. The remaining 75% of the EGF receptors displayed a marked tendency to exist as aggregates in nonionic detergent solutions.

Carcinoma, Squamous Cell↗

PubMed1980

Isolation of BPgp70, a fibroblast receptor for the envelope antigen of Rauscher murine leukemia virus.

A protein that avidly binds gp70, the envelope antigen of Rauscher murine leukemia virus (RMuLV), has been purified from the culture medium used for growth of BALB/c 3T3 mouse cells. Gel filtration chromatrography revealed the apparent Mr 10,000 BPgp70 was efficiently labeled when BALB/c 3T3 cells were grown in medium containing [3H]leucine, indicating a cellular origin for BPpg70. Metabolically labeled [3H]BPgp70 was not immunoprecipitated by IgG-anti RMuLV-gp&) alone, but was immunoprecipitated when gp70 was added, an indicaton of BPgp70 x gp70 complex formation. The dissociation constant estimated by immunoprecitipation agreed with the apparent Kd for binding of gp70 to BALB/c 3T3 cells. BPgp70 reversibly inhibited specific binding of 125I-labeled BMuLV-gp70 to BALB/c 3T3 cells when it was incubated with the 125I-labeled gp70 first. These data yielded a dissociation constant similar to that calculated from the immunoprecipitation data. 125I-Labeled BPgp70 also bound specifically to cells infected with RMuLV, but not to uninfected cells. Incubation of BALB/c 3T3 cells with the IgG fraction of an antiserum to BPgp70 inhibited the specific binding of 125I-labeled gp70 to these cells, but preimmune IgG did not. Complete inhibition was achieved at a less than 100:1 ratio of IgG anti-BPgp70 to gp70 binding sites.

Animals↗

PubMed1980

Protein-protein interactions within paramyxoviruses identified by native disulfide bonding or reversible chemical cross-linking.

Analysis of native disulfide-bonded protein oligomers in paramyxoviruses showed that some viral proteins are consistently present as covalent complexes. In isolated Sendai virus the hemagglutinating protein HN is present in homodimeric and homotetrameric forms, and the minor nucleocapsid protein P exists partly as a monomer and partly as a disulfide-linked homotrimer. Similar disulfide-linked complexes were observed in Newcastle disease virus (strain HP-16), in which HN exists as a homodimer and some of the major nucleocapsid protein NP exists as a homotrimer. Noncovalent intermolecular interactions between proteins were studied with the reversible chemical cross-linkers dimethyl-3,3'-dithiobispropionimidate and methyl 3-[(p-azidophenyl)dithio]propionimidate, which contain disulfide bridges and a 1.1-nm separation between their functional groups. The same results were achieved with both reagents. The conditions of preparation, isolation, and storage of the viruses affected the protein-protein interactions observed upon cross-linking. Homooligomers of the glycoprotein F, the matrix protein M, and the major nucleocapsid protein NP were produced in both Sendai and Newcastle disease viruses after mild cross-linking of all viral preparations examined, but NP-M heterodimer formation in both viruses was most prevalent in early harvest preparations that were cross-linked soon after isolation. The ability of NP and M to form a heterodimer upon cross-linking indicates that the matrix protein layer lies in close proximity (within 1.1 nm) to the nucleocapsid in the newly formed virion. Some noncovalent intermolecular protein interactions in Sendai and Newcastle disease viruses, i.e., those leading to the formation of F, NP, and M homooliogmers upon cross-linking, are more stable to virus storage than others, i.e., those leading to the formation of an NP-M heterodimer upon cross-linking. The storage-induced loss of the ability of NP and M to form a heterodimer is not accompanied by any apparent loss of infectivity. This indicates that some spacial relationships which form during virus assembly can alter after particle formation and are not essential for the ensuing stages of the infectious process.

Azides↗

PubMed1980

Protein organization in Newcastle disease virus as revealed by perturbant treatment.

Treatment of Newcastle disease virus with lithium diiodosalicylate differentially elutes the internally disposed proteins, M and NP, showing that these proteins are extrinsic, i.e., not associated with the lipid hydrophobic core. This selective elution requires disruption of the viral envelope, a process that is maximal at low temperature and influenced by the lipid composition of the virus envelope.

Electrophoresis, Polyacrylamide Gel↗

PubMed1980

Achromatic rorschach perceptions: some implications for the diagnosis of depression.

The number of Rorschach achromatic perceptions of 10 male and 10 female hospitalized uni-polar depressed patients were compared with a control group of 10 male and 10 female hospitalized patients who held diagnoses other than depression or mania. The depressed patients gave significantly more achromatic perceptions than the non-depressed group. There was also a significant effect for sex and in interaction. The long held belief, based on clinical observation, that achromatic perceptions distinguish depressed from non-depressed individuals, was supported by this study. However, the intricacies of the results indicate that direct application in clinical work may be premature without further clarifying research.

Adult↗

PubMed1980

Molecular identification and properties of two cell surface receptors playing roles in mitogenesis and epigenesis.

Studies leading to the identification of cell surface receptor macromolecules which specifically recognize a hormone, epidermal growth factor (EGF), or gp70, the coat antigen of the C-type RNA tumor viruses, have been described. EGF receptors are internalized and processed by lysosomal protease action after interaction of receptor and EGF. Other hormones, which resemble EGF in their ability to trigger mitogenesis in cultured cells, interact with the EGF receptor in a yet to be defined, but probably indirect way, decreasing the number of EGF binding sites on the cell surface. The evidence at this point indicates that these hormones interact with EGF receptors through the communal utilization of a shared cellular mechanism for internalization of their receptors and the EGF receptors. The receptor for gp70 is not internalized in response to gp70 binding, but is instead shed into the medium by cultured cells. This receptor protein BPgp70 has now been isolated and purified to apparent homogeneity. Antibodies to BPgp70 completely block gp70 binding to cells at low concentration, indicating that BPgp70 is the physiological receptor for gp70.

Animals↗