SELECTIVITY OF A C-TERMINAL PEPTIDE ANTISERUM FOR DIFFERENT DNA-BINDING STATES OF THE VITAMIN-D-RECEPTOR

Antisera against peptides from the extreme N- and C-terminal regions o f the VDR were evaluated, The N-terminal antiserum Ab192 functioned as a general-purpose antibody, being able to supershift the rhVDR in het erodimeric or homodimeric binding complexes in the ERISA, and detect b oth recombinant and native forms of the receptor on Western blots, The C-terminal antiserum, Ab195, also identified the receptor on Western blots, but in contrast, it displayed differential sensitivity to the c onditions employed in the EMSA. In the presence of 1,25(OH)(2)D-3, rhV DR, rhRXR alpha, and nonspecific DNA, Ab195 produced a weak supershift of the heterodimer complex in the EMSA, Significantly, omission of ho rmone from the binding buffer resulted in a complete shift of the boun d complex with the antiserum. A complete supershift was also observed if only the nonspecific DNA was removed, Together these results indica te antiserum sensitivity to the ligand status in the rhVDR C-terminus as pare of a DNA-bound heterodimer complex, Inclusion of 9-cis RA resu lted in a modest increase in the amount of shifted product relative to 1,25(OH)(2)D-3 alone, Finally, Ab195 completely supershifted the rhVD R homodimer binding complex under all tested conditions, including tho se analogous to where it was largely ineffective in shifting the heter odimer, Thus, Ab195 is sensitive to the ligand binding status of the V DR, discriminates heterodimer and homodimer binding interactions, and should provide an additional tool for exploring conformational changes induced in the receptor. (C) 1998 Academic Press.