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F. Felden, J. L. Guéant, A. Ennya, A. Gérard, S. Frémont, J. P. Nicolas, and H. Gérard


A specific receptor with high affinity for rat androgen-binding protein (rABP) was identified in isolated adult rat germ cells and in the corresponding plasma membrane-enriched preparations. Binding was reversible and time-dependent, with maximum relative binding after 40 min at 4 °C; it was pH-dependent, with maximum binding at pH 6–8. Unlabelled rABP and human sex steroid-binding protein (hSBP), but not lactotransferrin, serotransferrin, asialofetuin, fetuin or bovine serum albumin, competed with labelled rABP for binding sites on isolated germ cells. Scatchard analysis revealed a single class of binding site with apparent dissociation constant (K d) values of 0·78±0·04 nm and 0·97 ± 0·05 nm in intact germ cells and plasma membrane preparations respectively. A K d of 1·72±0·12 nm for hSBP showed that the receptor binding site was effective for both androgen-carrier molecules. Labelled rABP incubated with solubilized germ cell membrane fractions at pH 7 formed a complex excluded from Superose 6B mini-gels; this complex was not formed at pH 3. The receptor complex was also abolished in the presence of a 100fold excess of either unlabelled rABP or unlabelled hSBP, or in the presence of 20 mm EDTA.

These results suggest that the plasma membrane of rat germ cells contains a receptor which selectively binds rABP and hSBP.

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A. Gerard, M. Egloff, H. Gerard, A. El Harate, M. Domingo, J. L. Gueant, C. D. Dang, and H. Degrelle


Human sex steroid-binding protein (hSBP) has been purified from late-pregnancy serum and labelled either by iodination (125I) or by photoaffinity with [3H]Δ6-testosterone. Using a micromanipulator, each labelled protein was separately injected into the lumen of epididymal tubules isolated from the head epididymis of the cynomolgus monkey (Macaca fascicularis). Tubules were sampled from 3 to 90 min after the injection and processed for electron microscope autoradiography. Localization of the label occurred over the epididymal epithelium whichever tracer was used. The labelling was not randomly distributed over the different cell types constituting the epithelium, since only the 'principal cells' exhibited a silver grain count significantly greater than the background count. In these cells, labelled protein was found over endocytic organelles (coated structures, endosomes, multivesicular bodies and the trans Golgi network) and nuclei (including the nuclear envelope). Quantitative analysis demonstrated the same pattern of cellular and subcellular distribution for each tracer. Pretreatment with excess unlabelled protein significantly reduced the uptake of radioactivity by the principal cells, demonstrating the specificity of this phenomenon. This is the first study to show direct histological evidence for the internalization of hSBP in the primate epididymis, consistent with earlier immunohistochemical or biochemical localization of this protein. It is concluded that head epididymal cells are able to take up labelled hSBP across their apical membrane. The mechanism of internalization seems to involve endocytosis by the principal cells and leads to labelling of the nuclear compartment. This is strikingly similar to the pattern of uptake of rat androgen-binding protein (rABP) by rat epididymal cells previously demonstrated by our group. To what extent the chemical and structural homology between hSBP and rABP can be held responsible for the common cytophysiological behaviour of these sex steroid-binding proteins remains to be determined.

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J.-L. Guéant, S. Fremont, F. Felden, J.-P. Nicolas, A. Gerard, B. Leheup, H. Gerard, and G. Grignon


We have studied the binding of [ I-iodo]androgen-binding protein (ABP) and of [3H]Δ6-testosterone photoaffinity-labelled ABP to receptors in the plasma membrane of rat epididymal cells in three ways: ABP binding to a Triton X-100-solubilized membrane extract, ABP binding to isolated epithelial cells in suspension and autoradiography of segments of dissected epididymides after in-vitro intraluminal injection of labelled ABP. The binding of iodinated ABP to the receptor was similar to that of photoaffinity-labelled ABP in gel filtration. The ABP-receptor complex was eluted from Superose 6 gels as an aggregate, with a molecular mass of 2000 kDa. It was separated into two peaks by sucrose gradient ultracentrifugation, with respective sedimentation coefficients of 18.4 and 9.0s. The activity of the receptor (ABP-binding capacity/mg protein) was tenfold higher in the caput than in the cauda. The binding of ABP to the receptor was pH dependent, being almost abolished at pH <4. The binding at 4°C of photoaffinity-labelled ABP to epithelial cells corresponded to two types of binding sites. The numbers of high-affinity and low-affinity sites per cell were 1600 and 7700 respectively; the association constants of these sites were 67.9 and 2.8litres/ nm respectively. The binding was decreased by treatment of the cells with trypsin or incubation in the presence of ED TA. The binding in vitro of labelled ABP to the epididymis epithelium reached a maximum after about 20 min at 4°C. In the autoradiographic study the tracer was found to be closely associated with coated pits, coated vesicles, endosomes and pale multivesicular bodies. Treatment of rats with cycloheximide significantly reduced the uptake of the tracer. Perfusion in vitro of epididymides with chloroquine produced a fourfold increase of the tracer in endosomes and multivesicular bodies.

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Magda A Meester-Smoor, Anco C Molijn, Yixian Zhao, Nicole A Groen, Cora A H Groffen, Merel Boogaard, Diny van Dalsum-Verbiest, Gerard C Grosveld, and Ellen C Zwarthoff

The IGF-binding protein (IGFBP) family consists of six proteins that are expressed and secreted in different tissues. The proteins are regulators of physiological processes throughout the body by modulating the activity of IGF-I and IGF-II. In this article, we describe the coordinated expression of IGFBP5 and MN1 in meningiomas. MN1 is a transcriptional co-activator and we show that MN1 stimulates the IGFBP5 promoter in Hep3B cells. A CACCC-containing sequence, located 140 bp upstream of the transcription start site of the promoter, is required for MN1 action. This sequence matches with the CACCCAC consensus sequence that was selected in an oligonucleotide selection assay performed for MN1. The CACCC element has also been shown to be important for induction of the IGFBP5 promoter by retinoic acid (RA) and progesterone (Pg). We were unable to confirm the effect of Pg on the promoter in Hep3B and U2-osteosarcoma cells regardless of the presence of MN1. On the other hand, we show that induction of the promoter by RA depends on co-expressed MN1 in Hep3B cells. MN1TEL, a leukemia-related fusion protein containing parts of the MN1 and TEL (ETV6) genes, is capable of stimulating the IGFBP5 promoter but is unable to cooperate with RA in Hep3B cells. This suggests that the effects of RA can be negatively affected in leukemias caused by MN1TEL.