Publications

Abstract

The recent observation that adipose conversion of mouse 3T3 fibroblasts is stimulated by physiological concentrations of human GH (hGH) and rat GH in vitro suggested that this cell line may be suitable for the study of GH-receptor interactions. The aim of this study was to examine the binding and subsequent processing of [125I]iodo-hGH by BALB/c 3T3 mouse fibroblasts. Binding of [125I]iodo-hGH to 3T3 fibroblasts was time and temperature dependent. Apparent steady state binding was achieved after 1 and 2 h at 37 and 30 C, respectively. At 37, 30, and 20 C specifically bound [125I]iodo-hGH became increasingly resistant to removal by acid treatment (0.15 M NaCl/0.05 M glycine, pH 2.5). In contrast at 4 C or at higher temperatures in the presence of metabolic inhibitors, a greater proportion of specifically bound hGH was removed by acid treatment. Inclusion of 0.2 mM chloroquine in the incubation medium resulted in significantly more accumulation of trichloroacetic acid (TCA)-precipitable radioactivity compared to control cells without affecting the shift of radioactivity from the acid-elutable to the acid-inaccessible compartment. After removal of [125I]iodo-hGH from the medium there was a rapid loss of radioactivity (t 1/2 = 36.5 +/- 7.2 min, SE, n = 3) from the cell monolayer with a concomitant appearance in the medium of TCA-soluble radioactive species. Chloroquine reduced the rate of efflux of radioactivity from the monolayer (t 1/2 = 4.5 +/- 0.6 h, n = 3) and the appearance of TCA-soluble material in the medium. The half-time of GH receptor loss after inhibition of protein synthesis with cycloheximide (0.1 mM) was 1.25 +/- 0.14 h, n = 3). In contrast half-time of net receptor synthesis calculated from the recovery of specific [125I]iodo-hGH binding capacity after ligand-induced down-regulation was 10.2 +/- 1.5 h, n = 3). These data reveal that after binding of [125I]iodo-hGH to specific cell surface receptors there is rapid irreversible binding of GH to its receptor with a resultant reduction in receptor concentration. Degradation of [125I]iodo-hGH occurs intracellularly and involves processes which are inhibited by lysosomotropic agents. On the basis of these studies we conclude that the binding and subsequent processing of GH by 3T3 fibroblasts is qualitatively similar to that described for other polypeptide hormones and growth factors in this and other cell lines.