(11,12), suggesting a more than passive role of the IC domain in dimerization.The requirement for the EGFR tyrosine kinase activity in cellular signaling is based upon observations that receptors in which Lys-721 within the ATP binding site has been mutated and, hence, lack detectable kinase activity, do not display the full range of biochemical responses (13-15). This apparent requirement for kinase activity has focused attention on the development of drugs capable of blocking kinase activity specifically. Quinazoline inhibitors of the EGFR kinase are competitive with ATP; in the 1-10 nM range, they block EGFR phosphorylation and Src kinase activity in vivo but do not inhibit the platelet-derived growth factor receptor, p210Bcr-Abl , insulin receptor, CSF-1 receptor, and bFGF receptor tyrosine kinases (16 -19). In studying the ability of the EGFR kinase quinazoline inhibitors AG-1478 and AG-1517 to block TGF␣-induced signaling, their effect on receptor dimerization was measured. These studies demonstrated that quinazoline inhibitors per se induce inactive EGFR homodimers in EGFR-overexpressing cells or EGFR/ErbB-2 heterodimers in cells overexpressing ErbB-2 and containing lower levels of EGFR. The ability of the quinazolines to inhibit kinase function by sequestering receptors into inactive dimers appears related to their interaction with the receptor ATP binding site. These data suggest a novel biochemical mechanism of (inactive) receptor dimerization in which the initial monomer interactions are
The structural properties required for the binding of peptide substrates to the Escherichia coli periplasmic protein involved in oligopeptide transport were surveyed by measuring the ability of different peptides to compete for binding in an equilibrium dialysis assay with the tripeptide Ala-Phe-[3H]Gly. The protein specifically bound oligopeptides and failed to bind amino acids or dipeptides. Acetylation of the peptide amino terminus of (Ala)3 severely impaired binding, whereas esterification of the carboxyl terminus significantly reduced but did not completely eliminate binding. Peptides composed of L-amino acids competed more effectively than did peptides containing D-residues or glycine. Experiments with a series of alanyl peptide homologs demonstrated a decrease in competitive ability with increasing chain length beyond tripeptide. Competition studies with tripeptide homologs indicated that a wide variety of amino acyl side chains were tolerated by the periplasmic protein, but side-chain composition did affect binding. Fluorescence emission data suggested that this periplasmic protein possesses more than one substrate-binding site capable of distinguishing peptides on the basis of amino acyl side chains.
We describe a system for extending stopped-flow analysis to the kinetics of ligand capture and release by cell surface receptors in living cells. While most mammalian cell lines cannot survive the shear forces associated with turbulent stopped-flow mixing, we determined that a murine hematopoietic precursor cell line, 32D, is capable of surviving rapid mixing using flow rates as great as 4.0 mL/s, allowing rapid processes to be quantitated with dead times as short as 10 ms. 32D cells do not express any endogenous epidermal growth factor (EGF) receptor or other ErbB family members and were used to establish monoclonal cell lines stably expressing the EGF receptor. Association of fluorescein-labeled H22Y-murine EGF (F-EGF) to receptor-expressing 32D cells was observed by measuring time-dependent changes in fluorescence anisotropy following rapid mixing. Dissociation of F-EGF from EGF-receptor-expressing 32D cells was measured both by chase experiments using unlabeled mEGF and by experiments in which equilibrium was perturbed by dilution. Comparison of these dissociation experiments showed that little, if any, ligand-induced dissociation occurs in the chase dissociation experiments. Data from a series of association and dissociation experiments, performed at various concentrations of F-EGF in the nanomolar range and at multiple cell densities, were simultaneously analyzed using global analysis techniques and fit to a two independent receptor-class model. Our analysis is consistent with the presence of two distinct receptor populations having association rate constants of k(on1) = 8.6 x 10(6) M(-1) s(-1) and k(on2) = 2.4 x 10(6) M(-1) s(-1) and dissociation rate constants of k(off1) = 0.17 x 10(-2) s(-1) and k(off2) = 0.21 x 10(-2) s(-1). The magnitudes of these parameters suggest that under physiological conditions, in which cells are transiently exposed to nanomolar concentrations of ligand, ligand capture and release may function as the first line of regulation of the EGF receptor-induced signal transduction cascade.
The residue proposed to serve as the catalytic base for phosphoryl transfer, Asp-813, ofthe human epidermal growth factor receptor (EGFR) was mutated to Ala, and the mutant receptor (D813A) was expressed in Chinese hamster ovary (CHO) cells. Partially purified D813A exhibited no detectable kinase activity in the absence or presence of EGF. A low level of EGF-stimulable phosphorylation of D813A was detectable in intact cells, apparently due to the activity of an associated Tyr kinase(s). As previously observed for kinaseinactive Lys-721 mutants, EGF binding to D813A stimulates mitogen-activated protein kinase activity. Surprisingly, and unlike results reported for Lys-721 mutants, D813A is capable of stimulating both "Rb+ uptake and DNA synthesis in response to EGF. These data suggest not only that Asp-813 is critical to the catalytic activity of the EGFR but also that differences may exist in the signaling properties of kinasenegative Lys-721 and kinase-negative Asp-813 EGFR mutants.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.