Ligand-induced alteration of the protease sensitivity of retinoid X receptor alpha.

Ligand-induced structural alterations of retinoid X receptor (RXR) appear to facilitate receptor homodimerization, to enhance DNA binding of RXR homodimeric complexes, and to dictate the transcriptional activation properties of RXR complexes bound to response elements located in the promoter region of 9-cis-retinoic acid responsive genes. The technique of limited proteolysis was used to address 9-cis-retinoic acid-induced RXR conformational change and to identify receptor region(s) which undergo structural alteration upon ligand binding. Proteolytic digestion of 9-cis-retinoic acid-liganded, but not unliganded, RXR gave rise to a fragment of 31 kilodaltons (PF31), which contained a large portion of the RXR ligand binding domain. The potency with which 9-cis-retinoic acid induced formation of PF31 was nearly identical to that with which the ligand enhanced DNA binding of a RXR homodimeric complex to a response element composed of the directly repeated hexanucleotide, PuGGTCA, separated by 1 base pair. The amino-terminal limit of PF31 mapped to Ser229 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 binding which may directly alter the multiple functions of the RXR ligand binding domain.