Characterization of the Slowly Dissociable Human Growth Hormone Binding Component of Isolated Rat Hepatocytest

Human growth hormone (hGH) bound to specific sites on rat hepatocytes. The time course of hGH dissociation was comprised of more than one component. Dissociation was resolved into rapid ( t l l z = 10.5 min) and slow (tilz = 6.4 h) fractions. The amount of slowly dissociable hormone increased for the first 75 min during which time cells and [Iz5I]hGH associated. Subsequently, the amount of slowly dissociable hGH was constant. The time courses of hGH receptor binding and subsequent retention of slowly dissociable label were similar. The capacity of hepatocytes to accumulate slowly dissociable label was saturated by hGH over the same concentration range as the high-affinity binding site (KD = 2 nM). This suggested that a receptor-mediated process was responsible for the accumulation of slowly dissociable hGH. Rapidly M e c h a n i s t i c descriptions of peptide hormone-receptor binding and subsequent cellular response have assumed that bound hormone was free to dissociate rapidly to the medium (Cuatrecasas, 1974; Kahn, 1976). Isolated rat hepatocytes accumulate a slowly dissociable human growth hormone (hGH)' binding fraction with increasing incubation time (Donner et al., 1978a). Some characteristics of the accumulation and retention of insulin (unpublished experiments) 'From the Memorial Sloan-Kettering Cancer Center and Cornell University Graduate School of Medical Sciences, New York, New York 10021. Received September 19, 1979. Supported in part by Grants AM 19846, AM 22121, AM 15773, and CA 08748 from the National Institutes of Health. *Address correspondence to this author. D.B.D. is the recipient of a Research and Development Award from the American Diabetes Association. dissociable label was intact [ lZ5I] hGH and fragments resulting from growth hormone degradation. Slowly dissociable hGH recovered from hepatocytes by acid extraction was intact and immunocompetent. There was a large increase in the extent of [1251]hGH degradation between 23 and 37 "C. Over this temperature range, the proportion of hGH not in rapid equilibrium with the medium decreased. High concentrations of hGH decreased the amount of slowly dissociable ['251]hGH retained by hepatocytes by competing for high-affinity sites. The interaction of [1251] hGH with low-affinity degradative systems was favored by the presence of hGH. The temperature and concentration dependencies of hGH retention and degradation distinguished these processes. and glucagon (Martin et al., 1978a,b) are similar to those of hGH. This suggested that rapidly reversible equilibrium binding may not entirely describe peptide hormone-receptor interactions. Retention of intact, receptor-bound epidermal growth factor (Schecter et al., 1978) and thyrotropin (De Rubertis et al., 1975) was required for persistent cellular response to hormone. Therefore, a first step toward evaluating the ultimate significance of cellularly retained hGH was to determine whether this fraction of bound label was intact and receptor bound. The slowly dissociable hGH binding fraction on hepatocytes Abbreviations used: hGH, human growth hormone; ['*'I]hGH, iodine-1 25-labeled human growth hormone; HBSS, Hank's balanced salt solution; BSA, bovine serum albumin, fraction V; CI,AcOH, trichloroacetic acid. 0006-2960/80/0419-3293$01 .OO/O