Electric field-induced redistribution and postfield relaxation of epidermal growth factor receptors on A431 cells.

Abstract

Abstract. The lateral mobility of the epidermal growth factor (EGF) receptor in the plane of the plasma membrane of cultured A431 cells was investigated using direct and indirect fluorescent probes to measure the generation and relaxation of electric field-induced receptor asymmetry. A steady electric field of 15 V/cm for 30 min at 23°C induced a redistribution of the unoccupied EGF receptor such that there was approximately a three-fold higher concentration of receptors at the cathode-facing pole. After termination of the field, the unoccupied receptors back diffused at 37°C with a rate corresponding to a diffusion coefficient of 2.6-3.5 × 10 -l° cm2/s. No diffusion was detected at 4°C. Formation of the hormone-receptor complex is known to induce receptor clustering and internalization. By inhibiting internalization with metabolic poisons, we were able to study the cell surface mobility of clusters of the hormone-receptor complex. The same degree of asymmetry was induced when the occupied receptor was exposed to an electric field and the rate of back diffusion of clusters of the hormone-receptor complex corresponded to a diffusion coefficient of 0.68-0.95 × 10 -l° cm2/s. Although the unoccupied receptor is somewhat more mobile than the hormone-receptor complex, it was still far less mobile than one would predict for an unconstrained protein imbedded in a phospholipid bilayer.T o perform their physiological tasks, many integral membrane proteins must diffuse within the plane of the membrane to interact with other macromolecules. An accurate determination of the lateral diffusion rate of these proteins is important to understand these interactions. Most information gathered to date has used the technique of fluorescence photobleaching recovery (FPR) t to study cell surface receptors occupied with fluorescently labeled ligands. A small spot is bleached by an intense beam from a laser and the diffusion coefficient (D) is calculated from the rate of recovery of fluorescence as the unbleached receptor-ligand complex from adjacent areas diffuses into the bleached spot. D values determined by FPR for a number of receptors were in the order of 10-"-10 -l° cm2/s. These diffusion rates are ~100-fold slower than one might expect from theoretical calculations (Saffman and Delbruk, 1975;Saffman, 1976) and measured values for integral membrane proteins reconstituted into phospholipid bilayers (Peters, 1981;Tank et al., 1982). Artifacts of the FPR method have been considered as a possible explanation of this discrepancy, but, at least in certain model systems, the results of FPR experiments have been verified by independent methods (Koppel and Sheetz, 1981).Address reprint requests to Harry T. Haigler. One possible explanation for the unexpectedly slow D values determined in cells is that, by binding, the ligand induces interactions between its receptor and other cellular macromolecules that impede movement of the ligand-receptor complex. Unfortunately, the FPR method does not permit measurement of the D of unoccupied receptor...

Evaluation of EGFR and RTK Signaling in the Electrotaxis of Lung Adenocarcinoma Cells under Direct-Current Electric Field Stimulation

“…The EGFR pathway’s involvement in dcEF-cell interaction has been reported in epidermoid carcinoma cell A431 [62], bovine corneal epithelial cell [52,63], human keratinocyte [64,65], A549 lung adenocarcinoma cell [25], and MDA-MB-231 breast cancer cell [24]. Hepatocyte growth factor receptor (HGFR) has been reported to be related to the electrotaxis of bovine corneal epithelial cell [38].…”

Epidermal Growth Factor Receptor Distribution during Chemotactic Responses

“…However, contrary to the MTLn3 cells studied here, A431 cells have extremely high levels of EGFRs, a fraction of which might then be more stably associated with certain subcellular compartments. In keratinocytes polarized by electric fields, the receptor-ligand complex might be physically oriented by the electric field (Giugni et al, 1987), such redistribution being unnecessary in simple chemotactic gradients.…”

Role of Membrane Potential in the Regulation of Cell Proliferation and Differentiation

“…Membrane voltage potential [195,196] Activation of integrin or other signals by conformational changes in membrane proteins Membrane voltage potential [185,186,197] Depolarization-induced translocation of NRF-2 transcription factor Membrane voltage potential [198] Alteration of PTEN enzyme activity Membrane voltage potential [10,193,199] Redistribution of charged receptors along the cell surface Electric field [200][201][202][203][204][205] Directional electrophoresis of morphogens through cytoplasmic spaces Electric field [206,207] …”