FUNCTIONAL CONFORMATIONS OF THE NUCLEAR 1-ALPHA,25-DIHYDROXYVITAMIN D-3 RECEPTOR

The nuclear hormone 1 alpha,25-dihydroxyvitamin D-3 (VD) has important cell regulatory functions. Various synthetic VD analogues are under i nvestigation to identify candidates with an improved therapeutic profi le against hyperproliferative diseases. VD directly activates the tran scription factor VD receptor (VDR), which in turn stimulates the expre ssion of a cascade of primary and secondary VD-responsive genes. The a ctivation of the VDR through binding of its natural and synthetic liga nds is linked to a conformational change presenting the interface with co-activator proteins, referred to as the (trans)activation function 2 (AF-2) domain. Multiple conformations of the VDR might be the key to understanding a selective action of VD analogues. The method of limit ed protease digestion was used here to characterize up to three differ ent functional VDR conformations stabilized individually by VD and its analogues. The relative potency of VDR ligands can be quantified in t he interaction with these VDR conformations by determination of a func tional dissociation constant, where a two-concentration-point comparis on has already provided important information. In this way seven amino acid residues in the AF-2 domain have been analysed as potential liga nd contact points. Interestingly, residues Phe-422 and Val-418 seem to interact with all tested VDR ligands, whereas VD analogues such as th e anti-psoriatic drug MC903 displayed additional contact points within the AF-2 domain. Taken together, limited protease digestion is a powe rful method for studying functional VDR conformations and seems to be very appropriate for screening VD analogues.