LIGAND-INDUCED ALTERATION OF THE PROTEASE SENSITIVITY OF RETINOID-X RECEPTOR-ALPHA

Ligand-induced structural alterations of retinoid X receptor (RXR) app ear to facilitate receptor homodimerization, to enhance DNA binding of RXR homodimeric complexes, and to dictate the transcriptional activat ion properties of RXR complexes bound to response elements located in the promoter region of 9-cis-retinoic acid responsive genes. The techn ique of limited proteolysis was used to address 9-cis-retinoic acid-in duced RXR conformational change and to identify receptor region(s) whi ch undergo structural alteration upon ligand binding. Proteolytic dige stion of 9-cis-retinoic acid-liganded, but not unliganded, RXR gave ri se to a fragment of 31 kilodaltons (PF31), which contained a large por tion of the RXR ligand binding domain. The potency with which 9-cis-re tinoic acid induced formation of PE31 was nearly identical to that wit h which the ligand enhanced DNA binding of a RXR homodimeric complex t o a response element composed of the directly repeated hexanucleotide, PuGGTCA, separated by 1 base pair. The amino-terminal limit of PF31 m apped to Ser(229) of mouse RXR alpha, which lies roughly in the middle of the spacer region separating the DNA and ligand binding domains of the receptor. These results suggest that a fairly large region of the receptor protein undergoes a structural alteration upon ligand bindin g which may directly alter the multiple functions of the RXR ligand bi nding domain.