Intracellular potassium depletion in IM-9 lymphocytes suppresses the slowly dissociating component of human growth hormone binding and the down-regulation of its receptors but does not affect insulin receptors.

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We have investigated whether the slowly dissociating component of insulin and human growth hormone (hGH) binding and the homologous down-regulation of their receptors in IM-9 cultured human lymphocytes are due to distinct conformations of the receptor or, rather, to a redistribution within the cell. To do so, we used intracellular K+ depletion, which has been shown to inhibit reversibly coated-pit formation and ligand internalization in some cell lines. IM-9 cells incubated in K+-free buffer after a hypotonic shock rapidly lost their K+, which was stabilized at +/- 50% of control by incubation in K+-free binding assay buffer. In K+-depleted cells, the hGH dissociation kinetics became monoexponential and, in contrast with control cells, compatible with the equilibrium constant derived from saturation and association data using a simple model. The loss of hGH receptors during competition studies was abolished. The down-regulation by unlabeled hGH was decreased by 80%. In contrast, insulin receptor kinetics remained unchanged (non-first-order) in the K+-depleted cells; the negative cooperativity and the down-regulation (60%) were identical to those of control cells. Quantitative electron microscopic autoradiography showed a decrease of +/- 50% in the fraction of 125I-labeled hGH internalized. The number of visible coated pits in the membrane was reduced by 80%. Thus, in IM-9 cells, association with coated pits and endocytosis appear to play a major role in the kinetics of hGH binding and in the down-regulation of its receptors, but not in insulin-receptor binding kinetics and down-regulation.

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