Carol A. Donaldson

High pressure liquid chromatographic detection of intracellular retinoid binding proteins from cultured cell and tumor cytosols

We report the first application of high pressure liquid chromatography (HPLC) in the rapid detection of cellular retinoic acid binding protein (CRABP) and cellular retinol binding protein (CRBP). Cytosols from cultured cells (3T6 and MCF-7) or from tumors (melanoma and ovarian) were labeled with [3H]retinoic acid (30 Ci/mmol) and [3H]retinol (43 Ci/mmol) and analyzed via HPLC employing a 60 cm TSK 3000 sw column. In each case CRABP and CRBP were readily detectable at an elution volume of 22.5 ml, consistent with their molecular weights of 14,600. Identity of the binding protein peaks was established by saturability, specificity, and selective inhibition of binding by an organomercurial. Thus, this method, which resolves CRABP and CRBP in crude mixtures from the majority of cytosolic proteins, should be a valuable tool in the evaluation of vitamin A-binding protein interactions and their biological significance.

Identification and quantitation of intracellular retinol and retinoic acid binding proteins in cultured cells

Although the mechanism whereby vitamin A mediates normal cell differentiation and inhibits tumor cell proliferation is unknown, intracellular receptor-like proteins for retinol and retinoic acid have been implicated in the molecular action of vitamin A. We have assayed these two binding proteins, cellular retinol binding protein (protein R) and cellular retinoic acid binding protein (protein RA), in the cytosolic fraction of various normal and tumor cells via sucrose density gradient centrifugation and saturation analysis. Employing charcoal separation of bound and free tritiated retinoid, the saturation analysis yields an approximate Kd for ligand binding and an estimate of the number of protein R and protein RA molecules per cell. Unique protein R and protein RA macromolecules sedimenting at 2 S with Kd values of 7-42 nM are detected in murine cells (1 degree epidermal, 3T6 fibroblasts and melanoma) and human neuroblastoma cells. Concentrations of the intracellular binding proteins range from 55 000 to 3 000 000 copies per cell. When one cell line (C-127 mouse mammary) is transformed by bovine papilloma virus, protein RA levels increase from undetectable to 193 000 copies per cell. Assessment of growth inhibition by 10(-6) M retinol or retinoic acid in the culture medium reveals that there exists a partial, but not absolute, correlation between the presence of protein R or protein RA and the antiproliferative effect of the particular retinoid in the tested cell lines. We conclude that the 2 S intracellular binding proteins for the retinoids are present in most vitamin A responsive cells, but may not be essential for biologic actions of the vitamin such as growth inhibition in monolayer culture.

1,25-Dihydroxyvitamin D3 receptors: altered functional domains are associated with cellular resistance to vitamin D3.

1,25-Dihydroxyvitamin D3 receptors are cytosoluble proteins detectable in a variety of tissues responsive to 1,25(OH)2D3. They are DNA binding-proteins analogous to other steroid receptors and it is this functional property which is likely involved in the activation of hormone-sensitive genes. Utilizing 1,25(OH)2D3 and DNA binding assays, as well as anti-receptor monoclonal antibodies, we have probed the relationship between the 1,25(OH)2D3 receptor binding domains after selective cleavage with trypsin. These studies reveal that the hormone and DNA binding regions are separable, and are consistent with the finding that tissue resistance to 1,25(OH)2D3 is a result of structural defects in these domains. Recently, a primate model, the LLC-MK2 monkey kidney line, has been uncovered which may exemplify a hormone-binding defect. Here, 25-hydroxyvitamin D3-24-hydroxylase induction, a 1,25(OH)2D3 bioresponse, requires 100-fold higher concentrations of the hormone for maximal response. Concomitantly, this cell contains a variant receptor form which displays a correspondingly lowered apparent affinity for the hormone despite its seemingly normal DNA binding characteristics. Taken together, these studies suggest that the 1,25(OH)2D3 receptor is a macromolecule with multiple domains each of which may produce modified cellular resistance to 1,25(OH)2D3 if structurally altered.

Receptor-Mediated Action of the Vitamin D Hormone

1,25-Dihydroxyvitamin D3(1,25(OH)2D3) is now considered to be the active hormonal sterol derived from the sunlight vitamin, vitamin D3. As depicted in Fig. 1, 1,25(OH)2D3 is formed in the kidney according to the calcium and phosphorus needs of the organism (Haussler and McCain, 1977). Its main functions are the stimulation of intestinal calcium and phosphate absorption as well as bone remodeling. In addition to its mineral conservation effects in the kidney, 1,25(OH)2D3 induces a 24-OHase enzyme that appears to initiate a catabolic cascade for side chain cleavage and metabolic elimination of both the hormone (Fig. 1) and its 25(OH)D3 precursor (Chandler et al., 1984). Thus the 1,25(OH)2D3 hormone is dynamic in the sense that its production is controlled by calcium/parathyroid hormone (PTH) and phosphate status, and its biodegradation is self-initiated.