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Cytosolic free calcium ions concentration ([Ca2+]i) was measured in cell suspensions of cultured human IM-9 lymphocytes by dual wavelength fluorescence spectrometry using the calcium probe fura-2. Human GH (0.2-50 nM) induced a slow, progressive and sustained increase in [Ca2+]i. The GH effect was specific and exhibited a biphasic pattern, presumably reflecting GH receptor dimerization, typical of some other GH actions. The hGH effect depended on extracellular calcium, suggesting that at least part of the [Ca2+]i increase was due to a stimulation of calcium influx. GH did not increase IP3. Somatostatin-14 in the range 10(-10) to 10(-8) M, while having no effect of its own on [Ca2+]i, inhibited the effect of hGH. This inhibition by somatostatin was prevented by pretreatment of the cells with pertussis toxin. The hGH-induced [Ca2+]i increase was not related to either protein tyrosine phosphorylation or protein kinase C activation, thus suggesting a novel mechanism of GH transmembrane signalling.

Binding of GH to its cell surface receptors is thought to result in the formation of a complex comprised of one molecule of hormone per two molecules of receptor. It has been proposed that this hormone-induced receptor dimerization is important for the mechanism of signal transduction. We have developed a mathematical model for quantitative evaluation of the biological responses associated with sequential receptor dimerization. Based on these predictions, we have investigated whether GH-induced receptor dimerization plays a role in two classical effects of GH, i.e. stimulation of lipogenesis in primary rat adipocytes and GH receptor down-regulation in cultured human IM-9 lymphocytes. Model predictions of biological responses linked to dimer formation yielded a bell-shaped pattern, with self-antagonism at high GH concentrations when monomeric GH-receptor complexes become predominant. The GH lipogenic bioactivity curve was indeed biphasic and first increased in a concentration-dependent manner between 10(-10)-10(-8) M GH (ED50, 0.5 nM), up to a maximum of 1.7-fold stimulation above basal. Then, the response decreased continuously above 5 x 10(-8) M GH, returning to basal levels around 10(-5) M GH. Incubation of IM-9 cells with wild-type human GH resulted in a dose-dependent loss of their surface receptors. In contrast, a human GH analog (G120R), mutated in the second binding surface of the hormone and, therefore, unable to induce GH receptor dimerization, failed to induce receptor down-regulation in the IM-9 cells. Furthermore, when added together with wild-type human GH, human GH(G120R) inhibited, in a concentration-dependent manner, the down-regulation induced by wild-type human GH. Taken together, these data support the hypothesis that receptor dimerization is critical for the stimulation by GH of both lipogenesis in primary rat adipocytes and receptor down-regulation in cultured human IM-9 lymphocytes.

IM-9 cells extensively internalize [125I]human (h) GH at physiological temperatures, yet little is known regarding the final destination of internalized hormone and its receptor. We studied this by first binding [125I]hGH to the cell surface at 4 C, and then following its fate during a subsequent incubation at 30 C in isotope-free medium. Cell-associated radioactivity decreased with time at 30 C, with a biphasic pattern suggestive of a rapid (but minor) and a slow component. The kinetics of the latter were critically influenced by NH4Cl and were abolished at 20 C. Intracellular (acid-resistant) [125I]hGH first increased with time at 30 C until it reached a maximum after 1 h, then declined continuously upon prolonged incubation. The radioactivity released by the cells was recovered in the medium as both trichloroacetic acid-precipitable material and trichloroacetic acid-soluble fragments. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, one major band migrating with an estimated mol wt of 22,000 was identified, presumably corresponding to intact [125I]hGH. These data suggest exocytosis of intact hormone via recycling endosomes and degradation in the lysosomes, respectively. Computer modelling was consistent with two intracellular compartments acting partially in series and probably corresponding to these two organelles. When analyzed by computer curve fitting, this model accurately described the kinetics of [125I]hGH internalization. So, receptor-mediated endocytosis and subsequent exocytosis are part of the GH pathway in IM-9 cells. In as much as they reflect pathways of GH receptors, these processes contribute to receptor down-regulation and could provide an explanation for release into the medium of the high affinity GH-binding protein.

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.

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.