Comparison of the effects of hypertonic sucrose and intracellular potassium depletion on growth hormone receptor binding kinetics and down-regulation in IM-9 cells: evidence for a sequential block of receptor--mediated endocytosis

• Published in: Endocrinology (Philadelphia) Vol. 128; no. 3; p. 1597
• Main Authors: Ilondo, M M, Smal, J, de Meyts, P, Courtoy, P J
• Format: Journal Article
• Language: English
• Published: United States 01.03.1991
• Subjects: Cell Line; Down-Regulation; Endocytosis - physiology; Growth Hormone - metabolism; Humans; Hypertonic Solutions; Intracellular Membranes - metabolism; Kinetics; Lymphocytes - metabolism; Potassium Deficiency - metabolism; Receptors, Somatotropin - metabolism; Receptors, Somatotropin - physiology; Sucrose - pharmacology; Temperature; Time Factors
• ISSN: 0013-7227
• DOI: 10.1210/endo-128-3-1597

To better understand the complex kinetics of human GH (hGH) binding to its receptors, we have further investigated, in IM-9 cultured human lymphocytes, the cellular locus corresponding to the slowly dissociating component of hormone binding, and to the homologous down-regulation of hGH receptors. First, we have detailed the biphasic kinetics of dissociation of bound hormone in control cells at 30 C. When the association at 30 C was extended from 0.5 to 3 h, the time required for half-dissociation of the fast component was slightly decreased (from 30 to 15 min) but that of the slow component increased considerably (from 6 h to 30 h). Concomitantly, the size of the slowly dissociating component increased from 50 to 80% of total. This indicates a maturation of bound hormone, from a rapidly to a slowly dissociating pool and, in the latter, an increase in the apparent affinity that may reflect a molecular rearrangement. Next, we have compared the effect of two procedures reported to inhibit receptor-mediated endocytosis at the level of coated pits. As previously reported, depletion of intracellular K+ abolished the slowly dissociating component and the down-regulation of hGH receptors. In contrast, upon incubation with 0.4 M sucrose, which like K+ depletion virtually abrogated hGH internalization, the dissociation kinetics remained non-first order, and the down-regulation of hGH-receptor was only slightly reduced. Thus, these procedures appear to block receptor-mediated endocytosis at two successive compartments of the cell surface. In conclusion, we propose that some conformational change of hGH-receptor at the cell surface (possibly associated with clustering) may considerably slow down their dissociation and may be sufficient for down-regulation.