Lateral diffusion of epidermal growth factor complexed to its surface receptors does not account for the thermal sensitivity of patch formation and endocytosis

Abstract

The patching and endocytosis of EGF (epidermal growth factor) bound to A-431 cells (a human epidermoid carcinoma line) are temperature-sensitive processes which are completely inhibited at 4 degrees C. Receptor-mediated endocytosis generally occurs through coated regions, and EGF bound to its membrane receptor must diffuse laterally to these points of internalization. In this work we investigated the thermal sensitivity of the lateral diffusion of EGF receptor complexes and the thermal sensitivity of the patching and endocytosis of the hormone receptor complexes. Using the fluorescence photobleach recovery technique, we measured the lateral diffusion coefficients of a fluorescent derivative of EGF as a function of temperature. The lateral diffusion coefficient (D) increased gradually from 2.8 X 10(-10) cm2/s at 5 degrees C to 8.5 X 10(-10) cm2/s at 37 degrees C, and no phase transition was detected. Neither was a phase transition detected when we measured the diffusion coefficient of fluorescent lipid probes over this temperature range. From a calculation of the collision frequency of the occupied EGF receptors with coated regions using our measured values of D at 5 and 37 degrees C, we conclude that diffusion is not the rate-limiting step for either endocytosis or patching.