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.
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.
Ilondo
M M
MM
Department of Pediatrics, University of Leuven, Belgium.
Smal
J
J
de Meyts
P
P
Courtoy
P J
PJ
D002460:Cell Line; D015536:Down-Regulation; D004705:Endocytosis / Q000502:physiology*; D013006:Growth Hormone / Q000378:metabolism; D006801:Humans; D006982:Hypertonic Solutions; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D008214:Lymphocytes / Q000378:metabolism*; D011191:Potassium Deficiency / Q000378:metabolism*; D011986:Receptors, Somatotropin / Q000378:metabolism* / Q000502:physiology; D013395:Sucrose / Q000494:pharmacology*; D013696:Temperature; D013997:Time Factors
D006982:Hypertonic Solutions; D011986:Receptors, Somatotropin; D013395:Sucrose; D013006:Growth Hormone