Ligand- and DNA-induced dissociation of RXR tetramers

Abstract

Unliganded bacterially expressed RXRα lacking the N-terminal region AB (apo-RXRαΔAB) was found in solution as an apparent mixture of 165 kDa tetramers and 42 kDa monomers which could be quantitatively separated by gel filtration and non-denaturing gel electrophoresis. Under identical conditions both liganded (holo-) and apo-RARαΔAB were present as single monomeric species. apo-RXRαΔAB tetramers, as well as dimers of the apo-RXR ligand binding domain (apo-LBD), dissociated readily into monomers when exposed to their cognate ligand 9-cis retinoic acid (9c-RA). The apo-RXRαΔAB tetramer bound only transiently to a cognate DR1 response element, and was converted into DR1-apo-RXRαΔAB homodimer complexes indistinguishable from those generated by cooperative DNA binding of apo-RXRαΔAB monomers. In the absence of DNA, the addition of 9c-RA greatly accelerated the formation of hetero dimers with the apo-RARαΔAB heterodimerization partner. No RXRαΔAB or RARαΔAB homodimers could be observed in solution, but upon mixing of the two receptor monomers stable heterodimers could be isolated which bound to DR5 response elements in a highly cooperative manner. In these heterodimers, RXRαΔAB interacted with its cognate ligand as efficiently as in RXRαΔAB homodimers. The presence of ligand did not alter the stability of RXRαΔAB homodimer or RXRαΔAB-RARαΔAB heterodimer complexes on DR1 and DR5 response elements, respectively. These in vitro data support a model in which RXR tetramers could serve as an inactive pool with the dual function of: (i) rapidly supplying large amounts of RXR heterodimerization partners upon 9c-RA generation; and (ii) allowing RXR homodimer formation on 'accessible' cognate response elements in the absence of 9c-RA. These events may represent a ligand-dependent regulatory mechanism controlling the availability of the promiscuous RXR dimerization partner that is engaged in multiple nuclear receptor signalling pathways.