Characterization of the Ah receptor mediating aryl hydrocarbon hydroxylase induction in the human liver cell line Hep G2

The Ah receptor, a soluble cytoplasmic receptor that regulates induction of cytochrome P450IA1 and mediates toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was detected and characterized in the continuous human liver cell line Hep G2. The mean concentration of specific binding sites for TCDD was 112 +/- 26 (SEM) fmol/mg cytosol protein as determined in eight separate cytosol preparations in the presence of sodium molybdate. This is equivalent to 14,000 binding sites per cell, approximately 40% of the sites per cell found in the mouse hepatoma line Hepa-1. The cytosolic Ah receptor from Hep G2 cells sedimented at 9 S and was specific for those halogenated and nonhalogenated aromatic compounds known to be agonists for the Ah receptor in rodent tissues and cells. Specific binding in the 9 S region was detected with both [3H]TCDD and 3-[3H]methylcholanthrene. 3-[3H]Methylcholanthrene did not bind to any component besides that at approximately 9 S. Phenobarbital, dexamethasone, and estradiol did not compete with [3H]TCDD for binding to the Hep G2 Ah receptor. Specific binding of [3H]triamcinolone acetonide to glucocorticoid receptor could also be demonstrated in Hep G2 cytosol. The apparent equilibrium dissociation constant (Kd) for binding of [3H]TCDD to Hep G2 Ah receptor was 9 nM by Woolf plot analysis, about an order of magnitude weaker than the affinity of [3H]TCDD for the mouse Hepa-1 Ah receptor or for the C57BL/6 murine hepatic Ah receptor. [3H]TCDD.Ah receptor complex, which was extracted from nuclei of Hep G2 cells incubated with [3H]TCDD at 37 degrees C in culture, sedimented at approximately 6 S under conditions of high ionic strength. Aryl hydrocarbon hydroxylase (AHH) activity was significantly induced after 24 h of incubation with polycyclic aromatic hydrocarbons: the EC50 for AHH induction was 5.3 microM for benz(a)anthracene and 1.3 microM for 3-methylcholanthrene. Modification of the preparative technique for cell cytosol, especially inclusion of 20 mM sodium molybdate in homogenizing and other buffers, was necessary to detect cytosolic Hep G2 Ah receptor. Hep G2 cells appear to conserve drug-metabolizing activity associated with cytochrome P450IA1 as well as the receptor mechanism which regulates its induction.     

Structure and function of the Ah receptor: sulfhydryl groups required for binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to cytosolic receptor from rodent livers

Cytosol from rodent liver was exposed to a variety of sulfhydryl-modifying reagents to determine if the cytosolic Ah receptor contained reactive sulfhydryl groups that were essential for preservation of the receptor's ligand binding function. At a 2 mM concentration in rat liver cytosol, all sulfhydryl-modifying reagents tested (except iodoacetamide) both blocked binding of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to unoccupied receptor and caused release of [3H]TCDD from receptor sites that had been labeled with [3H]TCDD before exposure to the sulfhydryl-modifying reagent. Exposure of cytosol to iodoacetamide before labeling with [3H]TCDD prevented subsequent specific binding of [3H]TCDD, but iodoacetamide was not effective at displacing previously bound [3H]TCDD from the Ah receptor. The mercurial reagents, mersalyl, mercuric chloride, and p-hydroxymercuribenzoate, were more effective at releasing bound [3H]TCDD from previously labeled sites than were alkylating agents (iodoacetamide, N-ethylmaleimide) or the disulfide compound 5,5'-dithiobis(2-nitrobenzoate). Presence of bound [3H]TCDD substantially protected the Ah receptor against loss of ligand binding function when the cytosol was exposed to sulfhydryl-modifying reagents. This may indicate that the critical sulfhydryl groups lie in or near the ligand binding site on the receptor. Subtle differences exist between the Ah receptor and the receptors for steroid hormones in response to a spectrum of sulfhydryl-modifying reagents, but the Ah receptor clearly contains a sulfhydryl group (or groups) essential for maintaining the receptor in a state in which it can bind ligands specifically and with high affinity.     

1-amino-3,7,8-trichlorodibenzo-p-dioxin: a specific antagonist for TCDD-induced myelotoxicity

It was recently reported that suppression of murine bone marrow hematopoiesis is a very sensitive indicator for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity (1). We report here that a structural analog of TCDD, 1-NH2-3,7,8-trichlorodibenzo-p-dioxin (NH2-TriCDD), is a specific and effective antagonist for TCDD-induced myelotoxicity and enzyme induction. When administered to mice or added directly into culture at a 100-fold excess, relative to TCDD, NH2-TriCDD completely abrogated the ability of TCDD to inhibit granulocyte-macrophage progenitor cells (CFU-C) formation, an indicator of hematopoiesis. Further, NH2-TriCDD inhibited TCDD-induced activation of cytochrome P1-450 monooxygenase activity. Studies designed to measure specific binding of TCDD to the cytosolic Ah receptor indicated that NH2-TriCDD effectively inhibited binding of TCDD to the receptor by acting as a competitive antagonist (Ki = 0.72 nM).     

Androgen binding as evidenced by a whole cell assay system using cultured canine prostatic epithelial cells

The androgen receptor content in the prostate has been usually evaluated using subcellular fractions without taking into account cellular and functional heterogeneity of the gland. Using enriched populations of immature canine prostatic epithelial cells cultured in primary monolayers, a whole cell assay system was developed to measure androgen receptors. Tritiated dihydrotestosterone (DHT) and/or methyltrienolone (R1881) in serum-free medium were used as ligands and Triamcinolone acetonide (0.5 microM) was added to prevent the binding of R1881 to other types of receptors. The amount of radiolabelled ligand specifically bound to the cells was determined at equilibrium. Specific binding was proportional to the number of cells seeded. Scatchard analysis revealed the presence of at least two types of binding sites. The Kd for the high affinity binding site was 2 x 10(-9) M. Competition studies indicated that this component was specific for androgens; Methyltrienolone, Mibolerone and the antiandrogen RU 23908 were the most efficient competitors. They were followed by DHT, 5 alpha-androstane-3 alpha, 17 beta-diol, testosterone, estradiol and estrone. Progesterone, 5 alpha-androstane-3 beta, 17 beta-diol and epitestosterone were not inhibitors. The level of specific binding was 11.0 +/- 7.6 fmol of bound R1881 per 10(6) cells (n = 34) or 2075 +/- 1434 fmol per mg of DNA; these values correspond to an average of 6624 +/- 4577 sites per cell. Thus, using this whole cell assay system, specific and androgen receptors were detected in immature prostatic epithelial cells in culture. This assay will therefore be useful to study the interrelationship between androgen binding activity and specific cell functions.     

Membrane receptors for murine leukemia viruses: characterization using the purified viral envelope glycoprotein, gp71.

The 71,000 dalton glycoprotein (gp71) purified from Rauscher murine leukemia virus (R-MuLV) by affinity chromatography specifically binds to murine but not other mammalian cells in culture. Binding is prevented by specific antiserum raides to gp71 (anti-gp71). The binding assay as described in this report can detect receptors on as few as 300 murine cells, and with 1 X 10(5) cells gives significant binding with 30 sec. The results show that the purified glycoprotein retians biologic activity and can form a stable complex with specific receptors on mouse cell membranes. The assay can therefore be used to characterize the nature of the cellular receptors that are essential for leukemia virus infection. Purified gp71 binding to mouse cells is prevented if the cells are actively producing related ecotropic type C viruses, presumably because the receptors are occupied and are not available to bind exogenously applied gp71. The binding of gp71 to murine cells is enhanced by the presence of calcium ions and low pH. Binding studies performed using an excess of 125I-gp71 indicate the NIH/3T3 cells bind approximately 5.3 X 10(5) molecules of 125I-gp71 per cell.     

Lanthanide-based time-resolved fluorescence of in cyto ligand-receptor interactions

A lanthanide-based assay for ligand-receptor interactions provides an attractive alternative to the traditional radiolabeled determinations in terms of sensitivity, throughput, and biohazards. We designed and tested peptide ligands modified with an Eu-DTPA chelate. These labeled ligands were used in competitive binding assays with results comparable to those obtained using the traditional radiolabeled binding assays. The sensitivity of time-resolved fluorescence is sufficient to detect attomoles of europium, allowing assays in 96-well plates, compared with 30-mm dishes for (125)I binding assays to whole cells. We verified binding of Eu-DTPA-NDP-alpha-MSH to cells overexpressing the human melanocortin-4 receptor. The Eu-labeled ligand bound to these cells with an affinity similar to that of unlabeled NDP-alpha-MSH and was used to optimize a competitive binding assay. The lanthanide-based assays provided superior results with higher throughput and eliminated the need for radioactive waste disposal. This assay is appropriate for high-throughput screening of ligand libraries.     

Characterization of cell-surface receptors for monoclonal-nonspecific suppressor factor (MNSF)

Monoclonal-nonspecific suppressor factor (MNSF) is a lymphokine derived from murine T cell hybridoma. The target tissues are both LPS-stimulated B cells and Con A-stimulated T cells. Since the action of MNSF may be mediated by its binding to specific cell surface receptors, we characterized the mode of this binding. The purified MNSF was labeled with 125I, using the Bolton-Hunter reagent. The labeled MNSF bound specifically to a single class of receptor (300 receptors per cell) on mitogen-stimulated murine B cells or T cells with an affinity of 16 pM at 24 degrees C, in the presence of sodium azide. Competitive experiments showed that MNSF bound to the specific receptor and that the binding was not shared with IL2, IFN-gamma, and TNF. Various cell types were surveyed for the capacity to specifically bind 125I-MNSF. 125I-MNSF bound to MOPC-31C (a murine plasmacytoma line) and to EL4 (a murine T lymphoma line). The presence of specific binding correlates with the capacity of the cells to respond to MNSF. These data support the view that like other polypeptide hormones, the action of MNSF is mediated by specific cell surface membrane receptor protein. Identification of these receptors will provide insight into the apparently diverse activities of MNSF.     

Detection and characterization of the Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in the human colon adenocarcinoma cell line LS180.

The Ah (aromatic hydrocarbon) receptor mediates induction of aryl hydrocarbon hydroxylase (AHH; an enzyme activity associated with cytochrome P450IA1) by polycyclic aromatic hydrocarbon carcinogens such as 3-methylcholanthrene (MC) and benzo[a]pyrene (BP) and the halogenated toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Until recently the AhR seemed to be present only at very low levels in human cells and tissue. With a modified assay (the presence of sodium molybdate and a reduction in the amount of charcoal used to adsorb "excess" ligand) we found that cytosol from LS180 cells contains a high concentration of AhR (400-500 fmol/mg cytosolic protein) when detected by [3H]TCDD or [3H]MC. Cytosolic receptor also was detected with [3H]BP but at a level that was 35% of that detected with [3H]TCDD or [3H]MC. These levels are similar to those found in mouse Hepa-1 hepatoma cells in which AhR has been extensively characterized. The apparent binding affinity (Kd) of the cytosolic receptor for [3H]TCDD and for [3H]MC was about 5 nM. As with Hepa-1, the human LS180 cytosolic AhR sedimented at about 9 S on sucrose gradients when detected with [3H]TCDD, [3H]BP or [3H]MC. The nuclear-associated ligand.receptor complex recovered from cells incubated in culture with [3H]TCDD sedimented at about 6.2 S. The 9.8 S cytosolic form corresponds to a multimeric protein of a relative molecular mass (Mr) of about 285,000 whereas the 6.2 S nuclear receptor corresponds to a multimeric protein of Mr 175,000. The smallest specific ligand-binding subunit (detected by sodium dodecyl sulfate-polyacrylamide electrophoresis under denaturing conditions of receptor photoaffinity labeled with [3H]TCDD) was about Mr 110,000. AHH activity was induced in cells exposed in culture to TCDD or benz[a]anthracene (BA). The EC50 was 4 x 10(-10) M for TCDD and 1.5 x 10(-5) M for BA. For both inducers the EC50 in LS180 cells was shifted about one log unit to the right as compared to the EC50 for AHH induction in mouse Hepa-1 cells. The lower sensitivity of the LS180 cells to induction of AHH activity by TCDD or BA is consistent with the lower affinity of TCDD and MC for binding to human AhR. The ligand-binding properties, physicochemical properties, and mode of action of the AhR in this human cell line are therefore very similar to those of the extensively characterized AhR in rodent cells and tissues.     

Subunit structure of a laminin-binding integrin and localization of its binding site on laminin

A laminin receptor was isolated from human MG-63 osteosarcoma cells by affinity chromatography on human laminin. The isolated receptor was defined as the alpha 3 beta 1 integrin by immunoprecipitation with subunit-specific antibodies. A previously unclassified laminin-binding integrin from rat cells was shown also to contain the alpha 3 subunit. Both receptors bound to human and mouse laminin in a radioreceptor assay. They also both bound to some extent to fibronectin in this assay, but only the MG-63 cell receptor showed binding to type IV collagen. The binding of the radiolabeled receptor to insoluble laminin was inhibited by unlabeled receptor, by soluble laminin, and by chymotryptic fragments of laminin that have previously been shown to contain neurite-promoting and cell attachment-promoting activities. Moreover, the receptor binding was also inhibited by monoclonal antibodies capable of inhibiting the neurite-promoting activity of laminin and known to bind to laminin near the junction of the long arm and its terminal globule. One of these antibodies was reactive with fusion proteins expressed from laminin cDNA clones. The immunoreactive clones corresponded to the COOH-terminal end of the B1 subunit. These results identify the integrin-type laminin receptor isolated from the osteosarcoma cells as the alpha 3 beta 1 integrin and localize its binding site in close proximity of the B1 subunit COOH terminus.     

Molecular cloning and expression of the murine interleukin-5 receptor

Murine interleukin-5 (IL-5) is known to play an essential role in Ig production of B cells and proliferation and differentiation of eosinophils. Here, we have isolated cDNA clones encoding a murine IL-5 receptor by expression screening of a library prepared from a murine IL-5 dependent early B cell line. A cDNA library was expressed in COS7 cells and screened by panning with the use of anti-IL-5 receptor monoclonal antibodies. The deduced amino acid sequence analysis demonstrates that the receptor is a glycoprotein of 415 amino acids (Mr 45,284), including an N-terminal hydrophobic region (17 amino acids), a glycosylated extracellular domain (322 amino acids), a single transmembrane segment (22 amino acids) and a cytoplasmic tail (54 amino acids). COS7 cells transfected with the cDNA expressed a 60 kd protein that bound IL-5 with a single class of affinity (KD = 2-10 nM). FDC-P1 cells transfected with the cDNA for murine IL-5 receptor showed the expression of IL-5 binding sites with both low (KD = 6 nM) and high affinity (KD = 30 pM) and acquired responsiveness to IL-5 for proliferation, although parental FDC-P1 cells did not show any detectable IL-5 binding. In addition, several cDNA clones encoding soluble forms of the IL-5 receptor were isolated. Northern blot analysis showed that two species of mRNAs (5.0 kb and 5.8 kb) were detected in cell lines that display binding sites for murine IL-5. Homology search for the amino acid sequence of the IL-5 receptor reveals that the IL-5 receptor contains a common motif of a cytokine receptor family that is recently identified.     

Multiple binding components for methyltrienolone in canine prostatic epithelial cells

Using a whole cell assay system, the androgen binding capacity of canine prostatic epithelial cells was evaluated in relation to their function. Radiolabeled Methyltrienolone (R1881) was used as the ligand in the presence of an excess of Triamcinolone acetonide and the amount of [3H]R1881 bound to the cells at equilibrium was determined by either displacement or saturation studies. With immature cells in culture (3 days of attachment), displacement analysis revealed the presence of high affinity binding sites which were also present in cells cultured for 10 days. With freshly dispersed prostatic cells (mostly secretory epithelial cells) as well as with older cells in culture (17 and 24 days), only less specific binding sites were observed with both unlabeled R1881 and/or dihydrotesterone (DHT). In contrast, only the high affinity androgen receptor (AR) was present in cytosolic extracts prepared from normal glands. Displacement studies performed with cultured cells at different stages of growth also showed that the basal level as well as the degree of low affinity binding increased during the maturation of non-proliferating cells. The presence of multiple binding components was demonstrated by saturation studies performed with either cultured or freshly dispersed cells. The first component, that was saturated at 5 nM of [3H]R1881, was due to AR while the other two binding components, showing positive-cooperativity (Hill coefficients of 1.90 and 5.07, respectively), were saturated at concentrations of 15 and 30 nM of [3H]R1881. In contrast, the Hill coefficient for the AR was 0.88 indicating the presence of an independent component. It was calculated that only 11.4% of the total uptake of R1881 was attributed to AR binding, suggesting that the remainder may represent an intracellular pool of androgens. Thus, a whole cell binding assay represents a dynamic system for the detection, by saturation studies, of binding components that are not revealed using the conventional displacement studies or cell-free systems. It is proposed that these acceptor sites may play a role in differentiated prostatic function rather than in cell proliferation.     

Autoradiographic localization of aromatic hydrocarbon receptor (AHR) in rhesus monkey ovary

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic congener of a large class of manmade pollutants that persist in the environment. TCDD exerts its toxic effects, in part, by binding to its receptor known as the aromatic hydrocarbon receptor (AHR). TCDD is estrogen modulatory and in some systems its receptor associates directly with estrogen receptors via co-activator molecules. TCDD inhibits steroid synthesis in human ovarian granulosa cells and AHR is found in these cells. We have previously shown that AHR is found in whole rhesus monkey ovary, but have yet to establish its location. In the present study, we set out to show that radiolabeled TCDD binds to monkey ovarian follicles and that this binding is receptor mediated. Ovaries from Macaca mulatta were sectioned on a cryostat at 10 micro m; and sections were incubated with either control vehicle, (3)H-TCDD, or (3)H-TCDD plus alpha-naphthoflavone (ANF), a known receptor-blocking agent. Here, we show for the first time specific binding of TCDD to the granulosa cells of antral follicles and other regions of the rhesus monkey ovary. Our data indicate a 60-fold increase in binding with (3)H-TCDD over that of control, and that this binding is reduced to the levels seen in controls with the addition of the competitive antagonist ANF. These findings support the hypothesis that TCDD directly affects primate ovarian function via the AHR.     

Differences in 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible CYP1A1 expression in human breast carcinoma cell lines involve altered trans-acting factors

Differences in expression of the CYP1A1 gene have previously been observed in human breast carcinoma cell lines exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Using an expression vector containing the functional 5'-regulatory region of human CYP1A1 (up to -1140) fused to the reporter gene CAT (for chloramphenicol acetyltransferase), the breast carcinoma cell lines, MCF-7, T47-D and ZR-75-1, classified as highly responsive to TCDD, were highly responsive to TCDD in the chloramphenicol acetyltransferase assay as well. Gel mobility shift assays have shown that these cell lines express a nuclear protein that binds the aryl hydrocarbon (Ah) receptor responsive element. The low or non-responsive cell lines, AL-1, BT-20 and CAMA-1, were low or non-responsive to TCDD in the chloramphenicol acetyltransferase assay, suggesting that the low-responsive phenotype is caused by altered trans-acting factors. However, the mechanism appears to differ among the cell lines. Whereas no induction was observed in AL-1, a fivefold induction in activity was observed in BT-20 and CAMA-1. The TCDD concentration giving half-maximum induction differed greatly between CAMA-1 and BT-20. The gel mobility shift assay showed the presence of a protein that bound specifically to the Ah responsive element in the non-responsive cell line AL-1, as well as the low-responsive cell lines, BT-20 and CAMA-1. The high basal activity but low induction observed in CAMA-1 may be due to an Ah receptor constitutively bound to the Ah responsive element.     

Biological activity of 24,24-difluoro-1 alpha, 25-dihydroxyvitamin D3 and 1 alpha, 25-dihydroxyvitamin D3-26,23-lactone in inducing differentiation of human myeloid leukemia cells

Vitamin D compounds added to the culture medium induce differentiation of human myeloid leukemia cells (HL-60 cells) by binding to a specific cytosol receptor protein. This system provides a biologically relevant and technically simple assay to examine the relationship between molecular structure and biological activity of vitamin D compounds. Using this culture system, the biological activity of 24,24-F2-1 alpha,25(OH)2D3 and 1 alpha,25(OH)2D3-26,23-lactone was assayed. 24,24-F2-1 alpha,25(OH)2D3 was four to seven times more potent than 1 alpha,25(OH)2D3 in inducing phagocytosis and C3 rosette formation of HL-60 cells, though both compounds bound equally well to the cytosol receptor, suggesting that the defuorination at the 24-carbon position may stimulate membrane permeability of the compound. 1 alpha,25(OH)2D3-26,23-lactone, on the other hand, was only 1/200th as active as 1 alpha,25(OH)2D3. The binding affinity of the lactone for the cytosol receptor was identical with that of 1 alpha (OH)D3, suggesting that the lactone formation between the 26 and 23 positions masks the function of the 25-hydroxyl group. The binding affinity of vitamin D3 derivatives to the specific cytosol receptor of HL-60 cells was well correlated with that of intestinal cytosol protein specifically bound to 1 alpha,25(OH)2D3.     

Discrimination of a colony stimulating factor subclass by a specific receptor on a macrophage cell line

Utilizing the high affinity interactions between pure ¹²⁵I-L cell colony stimulating factor and its receptor(s) on the murine macrophage cell line J774, a murine radioreceptor assay (RRA) has been developed. The murine RRA selectively detects a colony stimulating factor (CSF) subclass (CSF-1) previously defined by murine radioimmunoassay (RIA) (E.R. Stanley, Proc. Nat. Acad. Sci., USA, 76:2969–2973 ('79)). CSF-1 stimulates macrophage production exclusively, and the occurrence of the CSF-1 receptor(s) appears to be restricted to cells of the mononuclear phagocytic system (L.J. Guilbert and E.R. Stanley, J. Cell Biol. 85:153–160 ('80)). The murine CSF-1 RRA failed to detect a variety of other CSF subclasses, growth factors, and hormones. In contrast to data obtained with the murine CSF-1 RIA, human CSF-1 (e.g., human urinary CSF) is detected by the mouse CSF-1 RRA almost as sensitively as murine CSF-1. In addition, there was an absolute correlation between CSF-1 levels determined by murine CSF-1 RRA and those determined by a human CSF-1 RIA for a variety of human CSF-1 sources. The murine CSF-1 RRA is a sensitive (sensitivity 5 units or 1.0 femtomole of CSF-1 protein), rapid, and highly specific assay for CSF-1 in both murine and human sources.     

Expression of recombinant MDA-BF-1 with a kinase recognition site and a 7-histidine tag for receptor binding and purification

Prostate cancer metastasizes predominantly to bone, where it induces osteoblastic lesions. Paracrine factors secreted by the metastatic cancer cells are thought to mediate these events. We previously isolated a novel bone metastasis-related factor (MDA-BF-1) from bone marrow aspirate samples from patients with prostate cancer and bone metastasis, and found that this factor stimulated osteoblast differentiation, possibly by interacting with a receptor on the osteoblasts. Identifying this putative MDA-BF-1 receptor biochemically requires the expression of MDA-BF-1 for receptor binding assays and for the preparation of a ligand-affinity column. We tagged MDA-BF-1 with a peptide containing a protein kinase A phosphorylation site plus a 7-histidine sequence to facilitate the labeling of MDA-BF-1 for receptor binding assay and the binding of MDA-BF-1 to an immobilized metal affinity column. The recombinant MDA-BF-1 protein (MDA-BF1-kinase-his) was expressed in Sf9 cells using a baculovirus expression system. About 0.8 mg of purified MDA-BF1-kinase-his protein was obtained from 4 x 10(8) Sf9 cells. MDA-BF1-kinase-his can be phosphorylated by PKA with a specific activity around 10(5)cpm/mug protein. Receptor binding assays using this (32)P-labeled MDA-BF-1 showed that MDA-BF-1 bound to membranes prepared from Saos-2, an osteosarcoma cell line, and C2C12, a mouse pluripotent mesenchymal precursor cell line that can be induced to become osteoblast by BMP-2. In contrast, MDA-BF-1 did not bind to membranes from PC-3 human prostate cancer cells or HEK293 human embryonic kidney cells. These observations suggest that the MDA-BF-1 receptor is expressed in cells of osteoblastic lineage. In addition to its use as a ligand for receptor binding assays, a ligand affinity column can be prepared by binding MDA-BF1-kinase-his to an IMAC for the purification of MDA-BF-1 receptor.