Characterization of Specific Growth Hormone Binding Sites in Mouse Fibroblasts*

Abstract

The demonstration that mouse embryo mesenchymal cells produce somatomedin-like immunoreactivity suggests that fibroblasts may be a target tissue for GH action. The aim of this study was to investigate the interaction of GH with mouse fibroblasts. Specific binding sites for GH in mouse fibroblasts (BALB/c and Swiss 3T3) have been characterized. Binding of [125I]iodo-human GH (hGH) was rapid, reversible, and time and temperature dependent. Maximal binding was achieved within 2 h at 30 C and was rapidly dissociable at this temperature with or without excess unlabeled hormone. Specific binding of [125I]iodo-hGH was similar over the pH range 6.6-8.2. Half-maximal inhibition of specific binding was obtained with 10 ng/ml hGH. A linear relationship between specific binding and cell number was found and negligible degradation of [125I]iodo-hGH occurred during the binding studies. Somatogenic hormones from various species, including mouse GH, rat GH, porcine GH, and bovine GH competed for binding with [125I]iodo-hGH. Lactogenic hormones did not displace [125I]iodo-hGH at low concentrations. Scatchard analysis revealed curvilinear plots suggesting that [125I]iodo-hGH was binding to two sites. The affinity constants and capacities of these binding sites on BALB/c 3T3 cells were 7.46 X 10(9) M-1 and 4,000 sites per cell and 0.26 X 10(9) M-1 and 67,000 sites per cell. Using [125I]iodo-bovine GH and [125I]iodo-human placental lactogen as labeled ligands two distinct binding sites were found with affinity constants of 6.1 X 10(9) M-1 and 1.1 X 10(9) M-1, respectively. These data are consistent with the presence of cell surface somatogenic and lactogenic receptors in mouse fibroblasts and suggest GH receptors may be present on many cell types not previously considered to be target tissues for GH action. The mouse fibroblast GH receptor may provide a useful model for somatogenic receptors, particularly if it is coupled to somatomedin production as appears to be the case with human fibroblasts.

Growth Hormone Binding to Specific Receptors Stimulates Growth and Function of Cloned Insulin- Producing Rat Insulinoma RIN-5AH Cells

“…A human lymphocyte cell line, IM-9 showed an apparent K d of 7.8 x 10" 10 M with 4,000 binding sites/cell (20). In these studies (18)(19)(20) Scatchard plots of the binding data were linear, while the binding of [ 125 I]hGH to mouse fibroblasts in monolayer cultures resulted in curvilinear Scatchard plots (21). The latter researchers, therefore, suggest the presence of two classes of binding sites with apparent K d values of 1.34 X 10" 10 and 3.8 X 10" 9 M and 4,000 and 67,000 binding sites/cell, respectively.…”

Changes in Levels of Serum and Urinary Collagen-Related Substances in Growth Hormone Deficient Children Following Discontinuation of Growth Hormone Therapy

“…Exogenous GH is considered to activate collagen metabolism in osteoblasts and osteoclasts and in the whole-body extracellular matrix. The action on collagen metabolism may be mostly mediated by insulin-like growth factor I, but also may be in part due to the action of GH itself on the liver or fibroblasts [6]. The ratio of levels of serum type III procollagen propeptide provides informations about patients' endogenous GH secretion, which may not be completely clarified by pharmacological provocation tests performed before receiving GH.…”

Effects of Hypophysectomy and Subsequent Growth Hormone Replacement in the Rat on the Ability of Serum to Stimulate Proliferation of Human and Mouse (BALB/c 3T3) Fibroblasts

“…All samples were assayed in the same assay. The sensitivity and within assay coefficient of variation of each of these assays in our laboratory have been previously reported (Murphy and Lazarus 1983).…”

“…BALB/c 3T3 cells were originally derived from the American Tissue Culture Collection, Rockville. Both cell lines were demonstrated to be free of mycoplasma contamination and were maintained in culture as previously described (Murphy and Lazarus 1983;Murphy, Vrhovsek and Lazarus 1983).…”

“…Thymidine Incorporation DNA synthesis in quiescent fibroblasts was determined by incorporation of tritiated thymidine into DNA as previously described (Murphy and Lazarus 1983). Triplicate wells were used for each serum concentration.…”