Dimerization of the Extracellular Domain of the Receptor for Epidermal Growth Factor Containing the Membrane-spanning Segment in Response to Treatment with Epidermal Growth Factor

Abstract

A recombinant fragment of the human receptor for epidermal growth factor containing both its extracellular domain and its membrane-spanning segment, when dissolved with Triton X-100, was observed to dimerize in response to addition of epidermal growth factor (EGF) even at the lowest concentration of this fragment that could be assayed (4 nM). Consequently, the dissociation constant for the dimer of this fragment is at least 10,000-fold smaller than that for dimers of soluble, recombinant forms of the extracellular domain lacking the membrane-spanning segment. The second-order rate constant for dimerization of the fragment containing the extracellular domain and the membrane-spanning segment was estimated to be greater than 0.3 nM ؊1 min ؊1 , more than 10-fold that of the native enzyme under the same conditions. This result suggests that the cytoplasmic domain of the intact enzyme sterically hinders its dimerization. When EGF is removed from the dimer of the fragment, the rate constant for its dissociation is greater than 0.2 min ؊1 , more than 40-fold that of the native enzyme. This result suggests that interfaces between cytoplasmic domains of intact EGF receptor impart significant stabilization to the dimer of the enzyme.Ligand-induced activation and dimerization of receptors in the plasma membranes of cells has received much attention (1-5). The dimerization of a monomeric form of the receptor for epidermal growth factor (EGF) 1 is an inescapable step (1) in the signal transduction initiated by this enzyme. The receptor for epidermal growth factor is composed of three domains (6): an extracellular domain that binds (7) the EGF, a membranespanning segment, and an intracellular domain responsible for its protein tyrosine kinase activity (8). It is of interest to determine the role of each of these domains in the dimerization of the intact protein.In the experiments reported here, a recombinant form of the extracellular domain of human EGF receptor also containing the membrane-spanning segment (9), dissolved in a solution of detergent, was assayed for its ability to undergo dimerization induced by EGF. Comparisons of the behavior of this extracellular domain of EGF receptor containing the membrane-spanning segment with that of the native, intact EGF receptor and with that of soluble, recombinant forms of the extracellular domain provide insight into the participation of the three domains in the process of dimerization. EXPERIMENTAL PROCEDURESPreparation of a Detergent Extract from B82 Cells-Cells of the murine B82 line expressing a recombinant form of EGF receptor that is missing its cytoplasmic domain and contains only the extracellular domain and the membrane-spanning segment (9) were graciously provided by Dr. Gordon Gill, Department of Medicine, University of California at San Diego. These cells were grown to confluence, the plates were scraped to release the cells, and the resulting suspension was spun at 3,000 rpm for 30 min in a Sorvall SS-34 rotor at 4°C. The pelleted cells were lysed (1) by the additi...

Role of Receptor Tyrosine Kinase Transmembrane Domains in Cell Signaling and Human Pathologies

“…The view that EGFR extracellular domains in the absence of ligands inhibit dimerization was first proposed by Tanner and Kyte, who carried out dimerization studies of the extracellular domain (50). Tanner and Kyte argued that the role of the ligand is to eliminate the steric repulsion between the extracellular domains.…”

“…In one study, aimed at elucidating the relative contributions of the extracellular domain and the TM domain, Tanner and Kyte showed that the extracellular EGFR domains dimerize strongly only in the presence of the TM domains (50). The dissociation constant of the extracellular domains, when attached to the TM domains, was estimated to be least 10,000 fold smaller, as compared to the dissociation constant of the isolated extracellular domains.…”

Structural Evidence for Loose Linkage between Ligand Binding and Kinase Activation in the Epidermal Growth Factor Receptor

“…The neu oncogene encodes a Val 3 Glu substitution in the TM domain of ErbB2 that results in constitutive activation (34). Recombinant EGFR fragments consisting of the extracellular and TM domains have a 10 5 -fold higher affinity for dimerization than the isolated soluble extracellular domains (31). The TM domains of all four ErbB family members self-associate when expressed in bacterial inner membranes (26).…”

Determination of the Dynamics, Structure, and Orientation of the Transmembrane Segment of ErbB2 in Model Membranes Using Solid-State NMR Spectroscopy

“…Further studies have also provided evidence that specific interactions between the TM helices of ErbB2 can mediate dimerization and receptor activation (8). In contrast to these findings, Tanner and coworkers have argued that interactions between the TM domains do not contribute significantly to ErbB2 receptor dimerization (9). Thus, the structural picture of the TM domain of ErbB2 remains elusive despite the extensive research devoted to this important protein.…”