Search PubMed⌕ Search

Biomedical subjects

A Sorkin

Publications and source records attributed to A Sorkin.

36 records · Page 2Linked to original sources
PubMed1996

Epidermal growth factor receptor interaction with clathrin adaptors is mediated by the Tyr974-containing internalization motif.

The carboxyl-terminal regulatory domain of the epidermal growth factor (EGF) receptor is essential for its endocytosis and interaction with the clathrin-associated protein complex AP-2. To identify AP-2 binding motif in the receptor, several single and multiple-point mutations within the region between residues 966 and 977 of the human EGF receptor were made, and the mutant receptors were expressed in NIH3T3 cells. Mutation of tyrosine 974 alone or together with surrounding residues and the deletion of residues 973-975 essentially eliminated AP-2 co-immunoprecipitation with the EGF receptor. Furthermore, a synthetic peptide corresponding to receptor residues 964-978 blocked AP-2 association with the wild-type EGF receptor. These data suggest that AP-2 has only one high-affinity binding site in the EGF receptor composed of Tyr974-containing motif. Receptor mutants that did not bind AP-2 displayed a lower rate of internalization, down-regulation, and turnover compared to wild-type receptors when expressed at high levels. However, similar receptor mutants expressed at low levels were internalized and down-regulated as efficiently as wild-type receptors. Internalization of the mutant receptors lacking the high-affinity binding site for AP-2 was inhibited by K+-depletion of the cells, indicating that their endocytosis required intact coated pits. We suggest that whereas one mechanism of EGF receptor recruitment into coated pits involves high-affinity binding of AP-2 to Tyr974-containing motif, another pathway may be mediated by weak receptor/AP-2 interactions or by proteins other than AP-2.

3T3 Cells↗
PubMed1995

Stoichiometric interaction of the epidermal growth factor receptor with the clathrin-associated protein complex AP-2.

Plasma membrane clathrin-associated protein complexes (AP-2) have been shown to co-immunoprecipitate with the epidermal growth factor (EGF) receptor (Sorkin A., and Carpenter, G. (1993) Science 261, 612-615). Hence, we analyzed the stoichiometry of the EGF receptor interaction with AP-2 using a new antibody that efficiently immunoprecipitates native AP-2. EGF receptor AP-2 complexes were isolated from 35S-labeled cells treated with EGF by EGF receptor affinity chromatography followed by precipitation with the antibody to AP-2. Quantitation of the relative molar concentrations of the proteins found in the complex revealed that 1 mol of AP-2 was associated with approximately 1.1 mol of EGF receptor. No other proteins were present in significant molar concentrations relative to AP-2, indicating that other proteins are not stoichiometrically involved in the interaction of EGF receptors and AP-2 in vivo. Co-immunoprecipitation experiments in cells expressing a mutant EGF receptor demonstrated that the cytoplasmic carboxyl-terminal 214 residues of the EGF receptor are essential for interaction with AP-2.

3T3 Cells↗
PubMed1994

Direct and indirect cost of percutaneous transluminal coronary angioplasty.

A prospective study of 53 patients employed in the 6-month period before coronary angioplasty was performed to determine the direct and indirect costs of lag time in work resumption. The total direct costs calculated were $273,480; indirect costs for this sample were $150,944. When these costs are generalized to all patients in the US undergoing uncomplicated percutaneous transluminal coronary angioplasty, the costs are more than $1.2 billion. This study demonstrated that even in patients with a high a priori probability of work return, delay in work resumption results in a greater cost to the individual and society through absence from the labor force.

Adult↗
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

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

Endocytosis of growth factor receptors.

Binding of a growth factor (GF) to its specific receptor on the cell surface causes the initiation of a signal transduction cascade which eventually results in mitosis. GF:receptor complexes are removed from the cell surface via receptor-mediated endocytosis, a process which involves clathrin-coated pits. After internalization into the endosomal compartment, a significant pool of GFs and GF receptors escape recycling to the cell surface and are sorted to the degradation pathway. The ligand-induced internalization and lysosomal degradation of GF receptors result in the dramatic loss of surface receptors, a phenomenon termed receptor down-regulation. In this review, we discuss relevant biochemical, morphological and kinetic studies of the mechanism of GF endocytosis, and the possible role of this process in mitogenic signaling by growth factor receptors.

Animals↗
PubMed1993

Pool of ligand-bound platelet-derived growth factor beta-receptors remain activated and tyrosine phosphorylated after internalization.

We have examined the state of tyrosine phosphorylation of ligand-bound, internalized platelet-derived growth factor (PDGF) beta-receptors. Analysis by immunofluorescence staining of cells stimulated with PDGF-BB at 37 degrees C indicated colocalization of phosphotyrosine, PDGF beta-receptors, and PDGF-BB in endosome-like vesicles. Treatment of cells with an acidic buffer, which removed cell surface-bound PDGF-BB, led to a considerable decrease in phosphorylation and kinase activity of cell surface localized PDGF beta-receptors, but not of internalized receptors. Immunoprecipitations using antisera against phosphotyrosine and the PDGF beta-receptor from metabolically labeled cells showed that a major fraction of the tyrosine-phosphorylated pool of receptors were still accessible to the acid buffer treatment after 10 min of incubation of the cells at 37 degrees C. Under these conditions, about 20-25% of the total pool of tyrosine-phosphorylated receptors were intracellular, since they remained tyrosine phosphorylated after the acid buffer treatment. A considerable pool of tyrosine-phosphorylated, internalized receptors, after 10 min of incubation of the cells at 37 degrees C, could also be detected by immunoblotting analysis, using antisera against the PDGF beta-receptor and phosphotyrosine. Analysis by in vitro kinase assays of immunoprecipitated PDGF beta-receptors, obtained from PDGF-BB-stimulated cells different times after acid wash, showed that the internalized receptors retained kinase activity. These data suggest that a pool of internalized PDGF beta-receptors remain active and may participate in signalling a considerable time after internalization.

Animals↗
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

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↗
PubMed1991

Dimerization of internalized epidermal growth factor receptors.

Binding of epidermal growth factor (EGF) to cell surface EGF receptors initiates the formation of the receptor homodimers that can be detected by covalent cross-linking in intact cells or in detergent-solubilized cell extracts. Low pH dissociation of EGF from surface receptors results in immediate monomerization of receptor dimers. Using chemical cross-linking during mild permeabilization or cell solubilization, we have detected dimers of internalized EGF receptors in human carcinoma A-431 cells and transfected NIH 3T3 cells that express human EGF receptors. The percentage of internalized cross-linked receptor dimers was similar to that observed for surface EGF receptors. Furthermore, at the time of maximal accumulation of EGF-receptor complexes within the endosomal compartment (10-15 min of incubation at 37 degrees C), both the dimeric and monomeric forms of the EGF receptor are tyrosine-phosphorylated to the same extent as surface dimer and monomer species. In transfected NIH 3T3 cells, the level of dimerized and internalized kinase-negative EGF receptors was not different from that observed for wild-type receptors. These data suggest that for some time after internalization EGF does not dissociate from its receptor and indicate that a receptor conformation is preserved intracellularly that allows maintenance of receptor-receptor interactions and tyrosine kinase activity.

3T3 Cells↗
PubMed1991

Multiple autophosphorylation site mutations of the epidermal growth factor receptor. Analysis of kinase activity and endocytosis.

We have utilized site-directed mutants to study the role of autophosphorylation of the epidermal growth factor (EGF) receptor in the regulation of receptor kinase activity and ligand-induced endocytosis. A single mutation of the major autophosphorylation site, Y1173, and a double mutation of two autophosphorylation sites, Y1173 and Y1148, did not inhibit kinase activity in vivo, using PLC gamma 1 as a specific substrate for the EGF receptor kinase. The simultaneous mutation of three major autophosphorylation sites (Y1173, Y1148, Y1068), however, caused more than a 50% decrease in EGF-induced tyrosine phosphorylation of PLC gamma 1. The triple mutation also resulted in a substantial inhibition of the EGF-receptor endocytic system. We have used three types of experiments to analyze internalization, recycling, and degradation of EGF in cells with these mutants or the wild-type receptor. Using a simple mathematical model we have shown that the internalization rate constant is 2-fold lower in cells expressing the triple mutation receptor (F3 cells) than in cells expressing wild-type EGF receptor (wild-type cells). However, the rate constant for recycling was similar in both cell types. The EGF degradation rate constant was also lower in F3 cells. EGF-induced EGF receptor degradation was slower in F3 cells (t1/2 = 4 h) than in wild-type cells (t1/2 = 1 h). Therefore, our results suggest that multiple autophosphorylations of the carboxyl terminus of the EGF receptor are required for EGF receptor kinase activation, and for the internalization and intracellular processing of the EGF.receptor complex.

Animals↗
PubMed1991

Recycling of epidermal growth factor-receptor complexes in A431 cells: identification of dual pathways.

The intracellular sorting of EGF-receptor complexes (EGF-RC) has been studied in human epidermoid carcinoma A431 cells. Recycling of EGF was found to occur rapidly after internalization at 37 degrees C. The initial rate of EGF recycling was reduced at 18 degrees C. A significant pool of internalized EGF was incapable of recycling at 18 degrees C but began to recycle when cells were warmed to 37 degrees C. The relative rate of EGF outflow at 37 degrees C from cells exposed to an 18 degrees C temperature block was slower (t1/2 approximately 20 min) than the rate from cells not exposed to a temperature block (t1/2 approximately 5-7 min). These data suggest that there might be both short- and long-time cycles of EGF recycling in A431 cells. Examination of the intracellular EGF-RC dissociation and dynamics of short- and long-time recycling indicated that EGF recycled as EGF-RC. Moreover, EGF receptors that were covalently labeled with a photoactivatable derivative of 125I-EGF recycled via the long-time pathway at a rate similar to that of 125I-EGF. Since EGF-RC degradation was also blocked at 18 degrees C, we propose that sorting to the lysosomal and long-time recycling pathway may occur after a highly temperature-sensitive step, presumably in the late endosomes.

Carcinoma, Squamous Cell↗
PubMed1991

Effect of receptor kinase inactivation on the rate of internalization and degradation of PDGF and the PDGF beta-receptor.

The complementary DNAs for wildtype and tyrosine kinase-inactivated (K634A) forms of the PDGF beta-receptor were expressed in porcine aortic endothelial cells. We examined the internalization and degradation of ligands and receptors after exposure of receptor expressing cells to PDGF-BB, which binds to the beta-receptor with high affinity, and PDGF-AB, which binds with lower affinity. Cells expressing wildtype beta-receptors were able to internalize and degrade the receptor, as well as the ligand, after exposure to PDGF-BB or -AB. Cells expressing the kinase-inactivated mutant receptor also internalized and degraded both receptor and ligand, but with lower efficiency compared with the wildtype receptor cells. The degradation of either form of receptor was inhibited by treatment of the cells with the lysosomotropic drug chloroquine. Exposure of wildtype and K634A receptor expressing cells to PDGF-AB resulted in a twofold slower rate of internalization of this ligand as compared with PDGF-BB, whereas the relative rate of degradation was similar for the two ligands. Our data indicate that tyrosine kinase activity promotes, but is not a prerequisite for, ligand-induced internalization and degradation of the ligand-receptor complex.

Animals↗
PubMed1989

Recycling of epidermal growth factor-receptor complexes in A431 cells.

The fate of epidermal growth factor (EGF) after internalization by human carcinoma A431 cells has been studied. Cells were allowed to internalize 125I-EGF for 10 min at 37 degrees C and treated with acid/salt solution to remove non-internalized ligand. Further incubation of these '125I-EGF-loaded' cells at 37 degrees C results in rapid recycling of internalized 125I-EGF-receptor complexes back to the cell surface. Recycling was assessed by measuring the increase in plasma membrane pool of 125I-EGF-receptor complexes as they became sensitive to acid/salt treatment, cross-linking with the membrane impermeant reagent bis(sulfosuccinimidyl)suberate and competitive substitution by unlabeled EGF. Moreover, redistribution of 125I-EGF-receptor complexes from endosomes to the plasma membrane was demonstrated using a subcellular fractionation technique. More than 50% of the total internalized EGF was found to be capable of recycling. The rate of recycling was significantly higher than that of EGF degradation in lysosomes. It was shown that EGF/receptor recycling is an energy-requiring and temperature-dependent process. Fluorescence microscopy studies demonstrate that endosomes located in a region adjacent to the Golgi complex are involved in the the recycling of EGF-receptor complexes in A431 cells. The data obtained suggest that dissociation of EGF from internalized receptor is not necessary for EGF receptor recycling.

Animals↗