Lactoferrin binding sites and nuclear localization in K562(S) cells.

Lactoferrin, a single chain cationic glycoprotein, present in the secondary granules of neutrophils, acts as a negative feedback regulator of myelopoiesis. Specific receptors for lactoferrin were detected on the surface of different hematopoietic cell types. The influence of lactoferrin on cell growth in culture has been reported. Interactions of lactoferrin with DNA were also demonstrated. In the present paper we confirm the presence of lactoferrin specific binding sites on K562 cells and we estimate the number of binding sites and the dissociation constant. By Western blotting analysis performed on K562 lysates we find a band of about 120 kDa responsible for specific binding of lactoferrin. We also show that lactoferrin, after binding at the cell surface, is internalized in a temperature dependent way and is immunologically detectable as a DNA-linked protein in nuclear extracts.     

Oestrogen and nuclear binding sites. Determination of specific sites by [3H]oestradiol exchange

A method was developed for the determination of the number of specific oestradiol-binding sites in the nuclear fraction of oestrogen-sensitive tissues. The method is based on the exchange of [(3)H]oestradiol with non-labelled oestradiol that is bound to nuclear binding sites. The number of specific nuclear binding sites after the injection of 2.5mug of oestradiol, an amount sufficient to saturate all binding sites, is 1.6-1.7pmol per immature uterus. The number of sites occupied after an injection of physiological amounts of oestradiol (0.1mug) was 0.46pmol. The injection of oestradiol results in an increased number of nuclear binding sites in uterus and vagina, but has no effect on kidney or muscle. Injections of testosterone or progesterone failed to increase the number of uterine nuclear binding sites. This method permits an evaluation of the number of oestradiol-binding sites in the nuclear fraction of various tissues as a function of either endogenous oestradiol or non-labelled oestradiol administered by injection.     

The identification of specific high density lipoprotein3 binding sites on human blood monocytes using fluorescence-labeled ligand.

We previously reported the identity and purification of two HDL3-binding proteins in rat liver plasma membranes. As these proteins are candidate high density lipoprotein (HDL) receptors and probably multifunctional, including a role in HDL metabolism, we have considerable interest in identifying corresponding proteins that are present in human tissue. This report describes the identification of HDL3-binding sites on human monocytes with the use of fluorescence microscopy and flow cytometry assay. After the incubation of mononuclear cells from human blood with fluorescein isothiocyanate (FITC)-labeled human HDL3, fluorescence micrographs showed dense signals of fluorescent grains on monocytes, but not lymphocytes. A significant increase in FITC intensity on monocytes, but not lymphocytes, was observed by flow cytometry analysis, and the interaction between FITC-HDL3 and human monocytes was concentration-dependent. Although very low density (VLDL) and low density lipoprotein (LDL) were ineffective competitors and HDL2 only partially competed for binding, a 50-fold concentration of HDL3 did compete effectively for binding of FITC-HDL3 to human monocytes. Trypsin treatment reduced the FITC intensity of monocytes, showing that a portion of cell-associated FITC-HDL3 remained bound to the cell surface. Two major HDL-binding proteins were identified in CHAPS-solubilized human mononuclear cells by ligand blotting, using HDL3 as the ligand. Both showed similar binding parameters, specificity, and molecular weight identical to HB1 and HB2 from rat liver plasma membrane. We conclude that corresponding candidate HDL receptors or a similar receptor complex also exist on human blood monocytes.     

Characterization of two high-density lipoprotein binding sites on porcine hepatocyte plasma membranes: contribution of scavenger receptor class B type I (SR-BI) to the low-affinity component

Two HDL(3) high- and low-affinity binding sites are present on the human hepatoma cell line (HepG(2)). Recently, we have suggested that the high-affinity binding sites might modulate the endocytosis of HDL through the low-affinity binding sites [Guendouzi, K. (1998) Biochemistry 37, 14974-14980], highlighting the physiological importance of this family of HDL high-affinity binding sites. The present data demonstrate the presence of HDL(3) high-affinity (K(d) = 0.37 microg/mL, B(max) = 260 ng/mg of protein) and low-affinity (K(d) = 86.2 microg/mL, B(max) = 14 300 ng/mg of protein) binding sites on purified porcine hepatocyte plasma membranes. By contrast, free apoA-I was strictly specific to the high-affinity sites (K(d) = 0.2 microg/mL and B(max) = 72 ng/mg of protein). Competition experiments between (125)I-labeled HDL(3) and either LDL, oxidized LDL, or anti-SR-BI IgG as competitors show that SR-BI is mostly responsible (70% displacement) for the binding of HDL(3) to the low-affinity binding sites. By contrast, the same competition experiments using (125)I-labeled free apoA-I clearly excluded SR-BI as the high-affinity binding receptor. We conclude that the binding of HDL onto hepatocyte plasma membranes involves: (1) two low-affinity binding receptors, one being SR-BI; (2) one family of high-affinity binding sites unrelated to SR-BI.     

The role of hepatocyte nuclear factor-3 alpha (Forkhead Box A1) and androgen receptor in transcriptional regulation of prostatic genes

Androgens and mesenchymal factors are essential extracellular signals for the development as well as the functional activity of the prostate epithelium. Little is known of the intraepithelial determinants that are involved in prostatic differentiation. Here we found that hepatocyte nuclear factor-3 alpha (HNF-3 alpha), an endoderm developmental factor, is essential for androgen receptor (AR)-mediated prostatic gene activation. Two HNF-3 cis-regulatory elements were identified in the rat probasin (PB) gene promoter, each immediately adjacent to an androgen response element. Remarkably, similar organization of HNF-3 and AR binding sites was observed in the prostate-specific antigen (PSA) gene core enhancer, suggesting a common functional mechanism. Mutations that disrupt these HNF-3 motifs significantly abolished the maximal androgen induction of PB and PSA activities. Overexpressing a mutant HNF-3 alpha deleted in the C-terminal region inhibited the androgen-induced promoter activity in LNCaP cells where endogenous HNF-3 alpha is expressed. Chromatin immunoprecipitation revealed in vivo that the occupancy of HNF-3 alpha on PSA enhancer can occur in an androgen-depleted condition, and before the recruitment of ligand-bound AR. A physical interaction of HNF-3 alpha and AR was detected through immunoprecipitation and confirmed by glutathione-S-transferase pull-down. This interaction is directly mediated through the DNA-binding domain/hinge region of AR and the forkhead domain of HNF-3 alpha. In addition, strong HNF-3 alpha expression, but not HNF-3 beta or HNF-3 gamma, is detected in both human and mouse prostatic epithelial cells where markers (PSA and PB) of differentiation are expressed. Taken together, these data support a model in which regulatory cues from the cell lineage and the extracellular environment coordinately establish the prostatic differentiated response.     

Studies on (NA + K)-activated ATPase XLV. Magnesium induces two low-affinity non-phosphorylating nucleotide binding sites per molecule

ATP and adenylylimidodiphosphate (AdoPP[NH]P) bind to (Na⁺ + K⁺)-ATPase in the absence of Mg²⁺ (EDTA present) with a homogeneous but 15-fold different affinity, the K_d values being 0.13 μM and 1.9 μM, respectively. The binding capacities of the two nucleotides are nearly equal and amount to 3.9 and 4 nmol/mg protein or 1.7 and 1.8 mol/mol (Na⁺ + K⁺)-ATPase, respectively. The K_d value for ATP is equal to the K_m for phosphorylation by ATP (0.05–0.25 μM) and the binding capacity is equivalent to the phosphorylation capacity of 1.8 mol/mol (Na⁺ + K⁺)-ATPase. Hence, the enzyme contains two high-affinity nucleotide binding and phosphorylating sites per molecule, or one per α-subunit. Additional low-affinity nucleotide binding sites are elicited in the presence of Mg²⁺, as shown by binding studies with the non-phosphorylating (AdoPP[NH]P). The K_d and binding capacity for AdoPP[NH]P at these sites is dependent on the Mg²⁺ concentration. The K_d increases from 0.06 mM at 0.5 mM Mg²⁺ to a maximum of 0.26 mM at 2 mM Mg²⁺ and the binding capacity from 1.5 nmol/mg protein at 0.5 mM Mg²⁺ to 3.3 nmol/mg protein at 4 mM Mg²⁺. Extrapolation of a double reciprocal plot of binding capacity vs. total Mg²⁺ concentration yields a maximal binding capacity at infinite Mg²⁺ concentration of 3.8 nmol/mg protein or 1.7 mol/mol (Na⁺ + K⁺)-ATPase. The K_d for Mg²⁺ at the sites, where it exerts this effect, is 0.8 mM. The K_d for the high-affinity sites increases from 1.5–1.9 μM in the absence of Mg²⁺ to a maximum of 4.2 μM at 2 mM Mg²⁺ concentration. The binding capacity of these sites (1.8 mol/mol enzyme) is independent of the Mg²⁺ concentration. Hence, Mg²⁺ induces two low-affinity non-phosphorylating nucleotide binding sites per molecule (Na⁺ + K⁺)-ATPase in addition to the two high-affinity, phosphorylating nucleotide binding sites.