Search PubMed⌕ Search

Biomedical subjects

J Schlessinger

Publications and source records attributed to J Schlessinger.

At least 361 records · Page 20Linked to original sources
PubMed1983

Regulation of cell proliferation by epidermal growth factor.

Epidermal Growth Factor (EGF) is a 6045 dalton polypeptide which stimulates the proliferation of various cell types in vitro and in vivo. EGF binds to diffusely distributed membrane receptors which rapidly cluster primarily on coated pits areas on the plasma membrane. Subsequently, the EGF-receptor complexes are endocytosed and degraded by lysosomal enzymes. The lateral diffusion coefficient (D) of EGF-receptor complexes on cultured cells increases gradually from D = 2.8 X 10(-10) cm2/sec at 5 degrees C to 8.5 X 10(-10) cm2/sec at 37 degrees C. In the same range of temperature the rotational correlation times change from 25 to 50 microseconds to approximately 350 microseconds. Hence, at 4 degrees C, the occupied EGF receptors translate and rotate rapidly in the plane of the membrane. At 37 degrees C, EGF receptors form microclusters composed of 10 to 50 molecules. Moreover, it is concluded that both at 4 degrees C and 37 degrees C lateral diffusion of the occupied receptors is not the rate determining step for either receptor clustering or internalization. EGF receptor is a 150,000 to 170,000 dalton glycoprotein. The receptor is in close proximity to an EGF-sensitive, cAMP-independent, tyrosine-specific protein kinase which also phosphorylates the receptor molecules itself. The EGF sensitive kinase is similar to the kinase activity which is associated with certain RNA tumor viruses. The fact that the non-mitogenic cyanogen-bromide cleaved EGF is as potent as native EGF in stimulating phosphorylation suggests that EGF-induced, protein phosphorylation is a necessary but insufficient signal for the induction of DNA synthesis by EGF. EGF receptor serves also as the binding site for Transforming Growth Factors (TGF) which compete with EGF and induce anchorage-independent growth of normal cells in soft agar. Tumor promoters such as phorbol ester effect the binding of EGF to its membrane receptors and its ability to stimulate DNA synthesis. EGF itself has also some tumor promoting activity. Hence, the membrane receptor for EGF seems to participate in the regulation of normal and neoplastic growth. Monoclonal antibodies against EGF receptor (IgM) induce various early and delayed effects of EGF, while their monovalent Fab' fragments are devoid of biological activity. These observations support the notions that EGF receptor rather than EGF itself is the active moiety and that the role of the hormone is to perturb the receptor in the appropriate way, probably by inducing the microaggregation of EGF receptors.

Allosteric Regulation↗
PubMed1982

Epidermal growth factor induces electrically silent Na+ influx in human fibroblasts.

Addition of epidermal growth factor (EGF) to quiescent confluent cultures of human foreskin fibroblasts causes a rapid, nearly 2-fold stimulation of unidirectional Na+ influx and a doubling of the rate of the Na+,K+ pump, whereas K+ efflux remains unaltered. The diuretic amiloride, an inhibitor of Na+/H+ exchange, completely blocks EGF-induced Na+ influx, Na+,K+-pump activity, and DNA synthesis without affecting the cellular binding, visible clustering, and internalization of 125I-labeled and fluorescent EGF. In the absence of EGF, the induction of amiloride-sensitive Na+ influx and Na+,K+-pump activity can be mimicked by exposing the cells to weak acids. Neither the rapid stimulation of Na+ influx by EGF nor its inhibition by amiloride is accompanied by a detectable change in membrane potential (mean value of -66 mV), as evidenced by direct intracellular recording. In contrast, a rapid but transient membrane depolarization of about 50 mV, due to an unselective permeability increase, is observed in response to serum-growth factors. These results (i) indicate that EGF rapidly activates an electroneutral, previously inactive Na+ transport system in the plasma membrane of quiescent fibroblasts, and (ii) suggest that EGF-induced Na+ influx occurs in exchange for intracellular protons. The data further imply that early changes in membrane potential are not necessary for the initiation of a mitogenic response.

Amiloride↗
PubMed1982

Lateral diffusion of epidermal growth factor complexed to its surface receptors does not account for the thermal sensitivity of patch formation and endocytosis.

The patching and endocytosis of EGF (epidermal growth factor) bound to A-431 cells (a human epidermoid carcinoma line) are temperature-sensitive processes which are completely inhibited at 4 degrees C. Receptor-mediated endocytosis generally occurs through coated regions, and EGF bound to its membrane receptor must diffuse laterally to these points of internalization. In this work we investigated the thermal sensitivity of the lateral diffusion of EGF receptor complexes and the thermal sensitivity of the patching and endocytosis of the hormone receptor complexes. Using the fluorescence photobleach recovery technique, we measured the lateral diffusion coefficients of a fluorescent derivative of EGF as a function of temperature. The lateral diffusion coefficient (D) increased gradually from 2.8 X 10(-10) cm2/s at 5 degrees C to 8.5 X 10(-10) cm2/s at 37 degrees C, and no phase transition was detected. Neither was a phase transition detected when we measured the diffusion coefficient of fluorescent lipid probes over this temperature range. From a calculation of the collision frequency of the occupied EGF receptors with coated regions using our measured values of D at 5 and 37 degrees C, we conclude that diffusion is not the rate-limiting step for either endocytosis or patching.

Animals↗
PubMed1982

Mobility of microinjected rhodamine actin within living chicken gizzard cells determined by fluorescence photobleaching recovery.

Rhodamine-labeled actin microinjected into living embryonic chicken gizzard cells became associated with its characteristic cytoskeletal structures. In these domains the translational diffusion coefficients (D) of rh-actin were determined in vivo by fluorescence photobleaching recovery (FPR) measurements. Two classes of actin molecules with respect to its mobilities were detected: rh-actin with a half-time of recovery of 5-10 min in stress fibers and focal contacts (immobile on the time-scale of FPR measurements) and rh-actin with D = 2-3 X 10(-9) cm2/sec in the cytoplasm and leading lamellae. The slow recovery on stress fibers exhibited similar kinetics whether a short segment or the entire structure were photobleached, indicating that recovery occurs predominantly by exchange with the surrounding diffusable actin. We propose that a steady-state equilibrium between the soluble and cytoskeletal pool of actin exists in living cells.

Actins↗
PubMed1982

Direct visualization of membrane clustering and endocytosis of thyrotropin into cultured thyroid cells.

We prepared a highly fluorescent conjugate of thyrotropin (TSH) which retains approximately 25% of the binding affinity of native TSH towards TSH receptor and approximately 25% of the potency of the native hormone in stimulating the accumulation of cAMP in thyroid cells. Using an image-intensified microscopy system, we observed that our fluorescent TSH bound specifically to diffusely distributed membrane receptors on live rat or bovine embryo thyroid cells grown in culture. At 37 degrees C the fluorescent hormone formed visible patches which were internalized and subsequently degraded. Hence, TSH, like other polypeptide hormones, is internalized by a process of receptor-mediated endocytosis. The addition of bivalent antibodies against the fluorophore rhodamine, to thyroid cells that were previously exposed to sub-optimal concentrations of rhodamine TSH, elicited maximal stimulation of the thyroid adenyl cyclase. Monovalent Fab' fragments did not enhance the response of rhodamine TSH. Therefore we conclude that enhanced surface clustering of TSH molecules increases their capacity to stimulate the adenyl cyclase of thyroid cells.

Animals↗
PubMed1982

A nonmitogenic analogue of epidermal growth factor induces early responses mediated by epidermal growth factor.

Cyanogen bromide-cleaved epidermal growth factor (CNBr-EGF) binds to EGF receptors with reduced affinity compared to the native hormone but fails to induce DNA synthesis. However, at similar receptor occupancy, CNBr-EGF is as potent as EGF in activating early cell responses to the hormone. The phosphorylation of membrane proteins, the stimulation of Na+-K+-ATPase as reflected by the ouabain-sensitive uptake of 86Rb of fibroblasts, changes in the organization of microfilaments and in cell-morphology, and the activation of the enzyme ornithine-decarboxylase are all induced by CNBr-EGF as well as EGF Our results are consistent with the notion that EGF-induced phosphorylation could act as a "second messenger" for the action of various EGF-induced responses such as activation of Na+-K+-ATPase, changes in the cytoskeleton and cell morphology, and the activation of the enzyme ornithine decarboxylase. However, the stimulation of phosphorylation of membrane proteins and other early responses are either not required or necessary but insufficient for the induction of DNA synthesis. Suboptimal concentrations of EGF together with CNBr-EGF stimulate DNA synthesis in human fibroblasts. Other growth factors such as insulin, fibroblast growth factor, and prostaglandin F2 alpha, which potentiate the mitogenic response of EGF, do not effect the response to CNBr-EGF. This suggests that the restoration of the mitogenic properties of CNBr-EGF by suboptimal doses of EGF occurs at the level of EGF receptors or during their processing.

Animals↗
PubMed1982

Restricted mobility of membrane constituents in cell-substrate focal contacts of chicken fibroblasts.

We studied the lateral mobility of membrane components in cell-substrate focal contacts using the fluorescence photobleaching recovery method. The measurements were performed on isolated substrate-attached membranes of chicken gizzard fibroblasts. The diffusion coefficients of a fluorescent lipid probe and rhodamine-conjugated surface proteins within contact regions (identified by interference-reflection microscopy) were significantly lower than those measured in nonattached areas along the ventral membrane. Complete recovery of fluorescence after photobleaching of the lipid probe was measured both in focal contacts and in nonattached areas with lateral diffusion coefficient (D) of approximately 10(-8) cm2/s. This indicated that the lipid probe is free to diffuse from and into the contact regions. Rhodamine-labeled surface components (mostly proteins) exhibited almost complete recovery after bleaching (approximately 90%) in unattached regions of the ventral membrane with D congruent to 10(-9 cm2/s. The rhodamine-labeled proteins in focal contacts showed only partial recovery (approximately 50%), suggesting that large proportion of the membrane proteins in cell-substrate contacts are immobile (within the time scale of the experiments, D less than or equal to 5 x 10(-12) cm2/s. The implications of these findings on the molecular dynamics of cell contacts are discussed.

Animals↗
PubMed1982

The interaction of cultured thyroid cells of early bovine embryos with thyrotropin and thyroglobulin.

We have prepared primary thyroid cell cultures of early bovine embryos from the first trimester of pregnancy in order to study the ontogeny of their interaction with TSH and thyroglobulin (Tg). The ability of these cells to synthesize and secrete Tg, as well as the trophic effect of TSH on the organization of the thyroid cells, were also investigated. To determine the maturation of these functions we prepared fluorescent conjugates of TSH, Tg, and anti-Tg antibodies, and visualized their interaction with the thyroid epithelial cells. Our study shows that the ability to bind TSH and Tg exists as early as the gestational age of 3 cm crown-rump length (CRL; 40 days) but does not develop linearly with embryonic age. Thus, there is a significant increase in the percentage of Tg-binding cells at 12 cm CRL, when colloid is first noticed in vivo, and a considerable elevation in TSH-binding cells around 15 cm CRL, when thyrotropic cells and TSH secretion from the fetal pituitary are first evident. Tg-containing cells and the ability to secrete Tg are observed at about 20 intrauterine days. The three thyroidal properties probably develop independently since only part of the Tg-containing cells bind Tg or TSH, and a significant proportion of the cells that exhibit Tg-binding do not bind TSH. The results support the notion that TSH is essential for the formation of follicle-like structures and effects the organization of thyroid cells into a functional structure in vitro from the late precolloidal stage.

Animals↗
PubMed1981

Adenosine 3',5'-monophosphate modulates thyrotropin receptor clustering and thyrotropin activity in culture.

A biologically active rhodamine conjugate of thyrotropin binds at 4 degrees C to diffusely distributed membrane thyrotropin receptors which patch and become endocytosed into thyroid cells in a temperature-sensitive process. When the cells are first incubated with 8-bromo-cyclic adenosine monophosphate at 37 degrees C, the conjugate also binds to clustered receptors at 4 degrees C. Furthermore, 8-bromo-cyclic adenosine monophosphate reduces the amount of adenosine 3',5'-monophosphate (cyclic AMP) induced by thyrotropin. Hence, increased intracellular cyclic AMP induces receptor patching and reduces the concentration of cyclic AMP normally induced by thyrotropin. This suggests that cyclic AMP acts both as the second messenger of thyrotropin and also as the regulator of the level of thyrotropin receptors.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1981

Primary amines do not prevent the endocytosis of epidermal growth factor into 3T3 fibroblasts.

Various amines block the degradation of endocytosed epidermal growth factor (EGF) without affecting the binding of the hormone to its surface receptors. However, studies based on fluorescence microscopy demonstrate that amines block the internalization of alpha 2-macroglobulin and EGF by preventing it from clustering in clathrin coated pits. In order to resolve this controversy we have studied in detail the effect of various amines on the localization and processing of fluorescent and radiolabelled EGF. We have explored the effect of amines on EGF binding and localization, receptor mobility, membrane fluidity, receptor down regulation, hormone degradation and release of degradative products as a function of time and temperature. Our conclusions are as follows. 1. Primary amines prevent the formation of visible patches of fluorescent EGF and alpha 2-macroglobulin on the cell surface at least for 15 min, thus increasing the diffusion coefficients and the mobile fraction of EGF-receptor complexes on the cell surface. 2. Amines do not block the endocytosis of EGF and alpha 2-macroglobulin. On most cells fluorescent EGF and alpha 2-macroglobulin are clustered and endocytosed within 30-45 min at 37 degrees C. 3. Amines do not effect the internalization of 125I-labelled-EGF and the down regulation of EGF receptors. 4. Amines block the degradation of the endocytosed EGF.

Acetates↗
PubMed1981

Rotational diffusion of epidermal growth factor complexed to cell surface receptors reflects rapid microaggregation and endocytosis of occupied receptors.

The rotational diffusion of epidermal growth factor (EGF)--receptor complexes on living human epidermoid carcinoma cells (A-431) has been measured by phosphorescence emission and anisotropy in the mu s time domain. A biologically active phosphorescent conjugate of EGF, erythrosin-EGF, was applied to living cells. The hormone--receptor complexes were mobile with rotational correlation times in the range 25--90 mu s when labeled and measured at 4 degrees C. Prolonged incubation and exposure to higher temperatures (23 and 37 degrees C) resulted in longer times up to 350 mu s, indicative of the progressive formation of microclusters, estimated to contain 10-50 receptors. Upon internalization of the hormone--receptor complexes, visible patches were observed by fluorescence microscopy, and the rotational correlation times were shorter, indicating a decrease in size of the dynamic unit. The sign of the rotational relaxation also varied with localization and processing of the hormones. The rate of lateral diffusion of EGF--receptor complexes, measured under similar conditions by fluorescence photobleaching recovery, increased with temperature in contrast to the rotational motion.

Animals↗
PubMed1981

Monoclonal antibodies against receptor for epidermal growth factor induce early and delayed effects of epidermal growth factor.

Mice were immunized with human epidermoid carcinoma cells (A-431 cell line) that possess an unusually high number of membrane receptors for epidermal growth factor (EGF). Spleen cells from these mice were fused with NSI cells, a nonsecreting murine myeloma. The immunoglobulins secreted by the obtained hybridomas were screened for specific binding to A-431 cells and selected according to their ability to inhibit the binding of radiolabeled EGF to the membrane of A-431 cells. Several antibodies secreted by cloned hybrid lines were found to inhibit the binding of radiolabeled EGF to membrane receptors of living A-431 cells, human foreskin fibroblasts, and mouse 3T3 fibroblasts and also to membrane preparations from A-431 cells. These monoclonal antibodies induced the early and delayed biological effects mediated by EGF. Like EGF, the antibodies induced morphological changes in A-431 cells and enhanced the phosphorylation of endogenous membrane proteins in membranes from these cells. They also stimulated DNA synthesis in human foreskin fibroblasts. These observations support the notion that the biological information of the EGF-receptor complex resides in the membrane receptor. Furthermore, the antibodies offer a powerful tool to study the structure, processing, and mode of action of EGF receptors.

Animals↗