Previous experiments from this laboratory have established the presence of receptors for insulin and insulin-like growth factor I (IGF-I) on apical membranes prepared from rabbit colon epithelial cells; however, no receptors for multiplication-stimulating activity (MSA), the rat peptide hormone equivalent of human IGF-II, were found in this tissue. In the current studies, radioligand binding assays, covalent cross-linking experiments, and immunoblot analyses using a polyclonal rabbit antiserum that recognizes the IGF-II/mannose 6-phosphate (Man-6-P) receptor, all confirmed the presence of IGF-II/Man-6-P receptors on membranes prepared from rat and human colon epithelial cells. Exposure of rat colon epithelial cell membrane fractions to 5 mM Man-6-P before incubation with 125I-labeled IGF-II increased radioligand binding. Immunoblot analysis indicated that IGF-II/Man-6-P receptors were present in both unfractionated rat colon membranes and fractions enriched with apical membranes. Rabbit and human colon epithelial cells displayed a different pattern of receptor distribution than rat colon epithelial cells, with more insulin receptors but relatively few IGF-II/Man-6-P receptors. Immunohistochemical studies using a rabbit polyclonal antiserum confirmed that IGF-II/Man-6-P receptors were present on both the apical and the basolateral surfaces of colon epithelial cells.
Factor(s) that bind gonadotropins have been extracted from rat testis by 30% ethanol (v/v) in water and their interaction with human lutropin (hLH) and human follitropin (hFSH) have been investigated by a new assay using dextran-coated charcoal.These studies reveal that:1. Maximal binding of gonadotropin with soluble factors was observed over a broad range of pH from 6.0 to 8.0 with a relative decline in binding at extremes of pH.The binding was independent of the ionic strength of the buffer and reached equilibrium within 5 min at 4", 27", and 37". 2. The soluble factors have marked thermostability, a point of distinction from detergent-solubilized receptors.3. The equilibrium dissociation constant (K,) of '%hFSH binding to the soluble factor was 6.0 * 0.58 x lo-l0 M, consistent with the values obtained from the membrane binding studies.Similarly, the Kd value for '*4-hLH to the soluble factor(s) was 3.33 l 0.3 x 10m9 M, comparable to the values obtained from the membrane binding studies.Hill plots demonstrated a lack of a cooperative relationship with an apparent Hill coefficient of 1.071 for hLH and 0.909 for hFSH.Furthermore, two classes of binding sites for '*51-human choriogonadotropin (hCG) were clearly discernible by both Lineweaver-Burk and Hill plots with an equilibrium dissociation constant of 2.4 + 0.5 x lo-" M and 1.35 * 1.2 x lo-' M. The apparent Hill coefficient of interaction of 'Y-hCG with the soluble factors was found to be 0.923 for high affinity and 1.09 for low affinity binding sites.4. The binding of '%hLH and **"I-hFSH with respect to concentrations of soluble factor(s) was found to be a saturable process, yielding an expected 4.4-fold higher K, for hLH (294 * 13.8 pg/ml) compared to hFSH (66.6 + 4 rg/ml).These findings are comparable with the equilibrium dissociation constants, thus confirming a 5-fold higher affinity of hFSH as compared to hLH for the soluble factors, i.e. the ratio of 3.0 x lo-' M to 6.0 x lo-l0 M versus the ratio of 294 pg/ml to 66.6 pg/ml.5. The hormone specificity of the interaction has been studied by using radiolabeled hFSH, hLH, hCG, prolactin, growth hormone, and bovine serum albumin.The binding of FSH at low factor concentrations was found to be 5. to lo-fold greater than prolactin, growth hormone, and albumin.6.The soluble factors are found in higher concentration in testis compared to liver, kidney, and blood.7. The effect of ethanol upon solubilization of the factor(s) has been investigated.The factor(s) can be extracted with buffer or water alone.However, 10 to 25%. of ethanol (v/v) facilitates the process of solubilization.The treatment with 70% ethanol (v/v) or more did not extract any factor activity from testes.The factor(s) were insoluble in petroleum ether, chloroform, absolute ethanol, methanol, or lipid solvent.8. Finally the effect of soluble factors on classical membrane binding was investigated.Increasing concentrations of the soluble factors resulted in graded inhibition of specific binding of '2JI-hLH and ""I-hFSH to fresh testicular homogenates.These studies implicate a possible regulatory role of soluble factors in hLH and hFSH activation of testis, more particularly as inhibitors of gonadotropin binding to testicular receptors.
Purified preparations of renal glomeruli and tubules were obtained by a procedure involving perfusion of rat kidneys with magnetic iron oxide particles to selectively separate the iron-containing glomeruli from the nonmagnetic tubules. Detergent-soluble extracts of both renal glomerular and tubular membranes showed high-affinity, specific binding of 125I-labeled insulin-like growth factor I (125I-IGF-I), whereas degradation of this peptide hormone was minimal during a 90-min incubation at 22 degrees C in the presence of 2.5 mM EDTA and 5 mM N-ethylmaleimide. The affinity of these receptors for IGF-I appeared identical in the two types of renal tissue, since 50% inhibition of 125I-IGF-I binding to both glomerular and tubular tissue occurred in the presence of approximately 3 x 10(-9) M unlabeled IGF-I. In contrast, insulin was much less effective at blocking 125I-IGF-I binding to either tissue, with 1 x 10(-6) M insulin required to produce 50% inhibition of binding. Relative to 125I-IGF-I binding, 125I-insulin binding to glomerular and tubular tissue was significantly lower per milligram protein. 125I-IGF-I was specifically cross-linked to a glomerular receptor subunit that migrated as two discrete bands with relative molecular weight (Mr) of 140,000-150,000 on sodium dodecyl sulfate polyacrylamide gels in the presence of 40 mM dithiothreitol. In contrast, 125I-IGF-I was cross-linked to a tubular receptor subunit that migrated as two discrete bands but at a slightly different position, with Mr of 120,000-140,000.(ABSTRACT TRUNCATED AT 250 WORDS)
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