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

Abstract

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.