Identification of the subdomain in the nuclear receptor for the hormonal form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3, vitamin D receptor, that is covalently modified by an affinity labeling reagent.

Multiple physiological actions of the hormonal form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3), are mediated by a genomic pathway which is initiated by the highly specific recognition and binding by its cognate receptor (vitamin D receptor, VDR) in the target cells. Thus, knowledge of the three-dimensional geometries of the ligand, i.e., 1,25(OH)2D3, and the 1,25(OH)2D3-binding domain of VDR is crucial for a better understanding of diverse physiological roles of this hormone. Recently our laboratory has developed 1 alpha,25-dihydroxyvitamin D3-3 beta-bromoacetate (1,25(OH)2 D3-3-BE) as an affinity labeling reagent for covalently modifying the hormone binding domain of native VDRs from calf thymus and rat osteosarcoma cells and baculovirus-expressed recombinant human VDR (hVDR). In the present report, we report affinity labeling of the hormone binding domain of hVDR, expressed in Escherichia coli as a glutathione S-transferase fusion partner, site-specific cleavage of the affinity-labeled VDR with 3-bromo-3-methyl-2-(2-nitrophenylmercapto)- 3H-indole, and identification of the C-terminal subdomain of human VDR containing the putative hormone binding site.

Identification of structurally altered estrogen receptors in human breast cancer by site-directed monoclonal antibodies.

We have developed and characterized site-directed monoclonal (MAb) and polyclonal antibodies to a specific domain in the N-terminal A/B region in order to assess estrogen receptor (ER) structural integrity in human breast tumor samples. The antibodies (Abs) reacted specifically with the native (undenatured) ER from various species. The synthetic peptides competed effectively for ER binding to the Abs, suggesting site-specificity. The Abs recognized the activated (4S) and transformed (5S) but not the unactivated, untransformed, molybdate-stabilized (8S) ER, suggesting that the epitope is inaccessible in the 8S form. Some of these Abs reacted with ER bound to its responsive elements, as determined by gel mobility shift assay. To evaluate the structural integrity of ER in breast cancer, we have utilized a) ligand binding analysis for the hormone binding domain; b) site-directed MAb to the DNA-binding domain; and c) site-directed MAb to the N-terminal transactivation domain. Analysis of ER from 29 human breast tumors revealed that 10 out of 29 tumors (35%) contained ER with intact hormone-, DNA-, and N-terminal domains. Thirteen out of 29 tumors (approximately 45%) contained ER with intact hormone binding and N-terminal domains but were defective only in the DNA-binding domain. Three out of 29 tumors (approximately 10%) contained ER defective only in the N-terminal domain. Another subgroup of tumors (3/29; approximately 10%) had ER with normal hormone binding domain but were defective in both the DNA-binding and the N-terminal activation domains.(ABSTRACT TRUNCATED AT 250 WORDS)