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A small group of closely related proteins is responsible for all active transport in animal cells, and inorganic cations are the only substances transported by these enzymes. They share a common kinetic mechanism in which two fundamental conformations participate, each receiving and dispatching substrates from its unique side of the membrane. During transport, the cations must pass through their enzyme to cross the membrane and intense interest is currently focused on the possibility that the path which they follow lies within the interface between two discrete subunits in a dimeric structure. Although 'half-of-sites' behaviour, consistent with this hypothesis, has been reported, it is now known that systematic errors were responsible for this mistaken conclusion. The number of protomers which comprise a functional unit of active transport has not been determined.

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...

The binding of epidermal growth factor (EGF) to epidermal growth factor receptor (EGF receptor) induces dimerization of the receptor and activation of its protein tyrosine kinase. Each of these three steps was followed as a function of the concentrations of EGF and of EGF receptor. Binding of EGF was followed by sedimentation of the complex between [3H]EGF and EGF receptor, dimerization was measured by quantitative cross-linking with glutaraldehyde, and the activation of the protein tyrosine kinase was monitored under the same conditions by following the initial velocity of the phosphorylation of peptides containing tyrosine. The binding of epidermal growth factor to its receptor was measured as a function of the concentration of epidermal growth factor, and the relationship was sigmoid with an average value of 1.7 for the Hill coefficient. Both dimerization and the activation of the tyrosine kinase displayed saturation as a function of the concentration of EGF. The ranges of the concentrations of EGF where dimerization and activation of the tyrosine kinase activity were half-maximal were 15-30 and 50-200 nM, respectively, but the value for the concentration of EGF at the half-maximum for the activation of the tyrosine kinase was a complex function of the concentration of EGF receptor. The observed behavior of the binding of EGF, the dimerization of EGF receptor, and the activation of the tyrosine kinase were used as criteria against which to test mechanisms for the process of activation. Equations were derived for various reversible and irreversible mechanisms and used to calculate the theoretical behaviors of the three properties. In direct comparisons of the experimental and the theoretical data, several of the previously proposed reversible and irreversible mechanisms for the activation of EGF receptor were found to be inadequate, but a reasonable mechanism was formulated that was compatible with the experimental data. In this mechanism, dimeric EGF receptor must be occupied by two molecules of EGF for enzymatic activity to be expressed.