Direct visualization of binding, aggregation, and internalization of insulin and epidermal growth factor on living fibroblastic cells

We have studied in detail the binding of fluorescent derivatives of insulin and epidermal growth factor to 3T3 fibroblasts. We have used two types of fluorescent analogues of insulin and epidermal growth factor:highly fluorescent derivatives which have seven to eight rhodamine molecules orfluorescent derivatives which have a single rhodamine molecule per one molecule of insulin or epidermal growth factor. Both types of analogue retained substantial binding affinity as determined by radioreceptor assays and biological activity. The cells labeled with the fluorescent analogues were visualized with a sensitive video intensification microscopic system that enabled us to directly observe the location of the fluorescent hormone on the surface and within the living fibroblasts. We found that both insulin and epidermal growth factor initially bound diffusely to the cell surface and, at 4°, remained dispersed. Within a few minutes at 23° or 37° the hormone-receptor complexes aggregated into patches that could be readily removed by trypsin but not by excess native hormone. The hormone-receptor complexes, which were initiallymobile in the plane of the membrane, become immobilized later as the consequence of the receptor aggregation or internalization. Within ∼30 min at 37°, much of the labeled hormone was found within the cell in endocytic vesicles that moved about in the cytoplasm in a saltatory manner. The aggregation and immobilization of the hormone-receptor complexes could be due to either hormone-hormone interactions on the cell membrane or a hormone-induced conformational change in the hormone-receptor complex. Aggregation and internalization of hormone-receptor complexes could be associated with certain aspects of hormone action, hormone degradation, down regulation of receptors, or negative cooperativity of hormone binding.