Initial reports of the interaction between human GH (hGH) and IM-9 cells described rapid and reversible binding to membrane receptors. In this study we present further investigations of hGH binding to IM-9 cells and suggest that this interaction does not demonstrate equilibrium kinetics but, rather, involves compartmentalization of both hormone and receptor. Incubation of IM-9 cells with [l25I]hGH failed to demonstrate steady state binding. After 8 h at 30 C, specific binding was 100% greater than at 90 min (P < 0.001), with similar findings following incubation at 37, 15, and 4 C. As early as 15 min of incubation, the addition of excess unlabeled hGH for 1 h failed to dissociate over 50% of specifically bound hormone. The nondissociable fraction increased as a function of incubation time, so that by 6 h of incubation, 80% of labeled hormone could not be dissociated by 1 h of exposure to excess unlabeled hGH. Although more prolonged exposure to unlabeled hGH resulted in slightly greater dissociation of cell-bound ['"'I]hGH, the total percentage of [l25I]hGH which was both specifically bound and nondissociable increased as a function of the duration of association. Similarly, after 30 min of incubation, trypsinization of the cells failed to dissociate 50% of bound hormone, with a timedependent increase in the trypsin-resistant fraction. Incubation of IM-9 cells with [l25I]hGH and 10 4 M cycloheximide failed to alter the nondissociability of bound hGH, suggesting that new protein synthesis is not required for compartmentalization. When cells incubated with [l25I]hGH for 2-6 h were solubilized in 1 M acetic acid and chromatographed over Sephadex G-75, over 70% of the cell-bound radioactivity eluted in the region of intact hGH. The data presented support the concept that membranebound hGH does not remain in equilibrium with the extracellular environment but is rapidly compartmentalized in a manner causing the hormone to become relatively inaccessible to dissociating agents. In view of these findings, the concept of hormonereceptor binding as a totally reversible association between ligand and membrane receptor sites, requires reevaluation.
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism
- Clinical Biochemistry
- Biochemistry, medical