Functional conformations of the nuclear 1alpha,25-dihydroxyvitamin D3 receptor.

The nuclear hormone 1alpha,25-dihydroxyvitamin D3 (VD) has important cell regulatory functions. Various synthetic VD analogues are under investigation to identify candidates with an improved therapeutic profile against hyperproliferative diseases. VD directly activates the transcription factor VD receptor (VDR), which in turn stimulates the expression of a cascade of primary and secondary VD-responsive genes. The activation of the VDR through binding of its natural and synthetic ligands 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 limited protease digestion was used here to characterize up to three different functional VDR conformations stabilized individually by VD and its analogues. The relative potency of VDR ligands can be quantified in the interaction with these VDR conformations by determination of a functional dissociation constant, where a two-concentration-point comparison has already provided important information. In this way seven amino acid residues in the AF-2 domain have been analysed as potential ligand contact points. Interestingly, residues Phe-422 and Val-418 seem to interact with all tested VDR ligands, whereas VD analogues such as the anti-psoriatic drug MC903 displayed additional contact points within the AF-2 domain. Taken together, limited protease digestion is a powerful method for studying functional VDR conformations and seems to be very appropriate for screening VD analogues.