Analysis of Homo- and Heterodimerization of Retinoid Receptors in Solution

To characterize the dimerization of retinoid receptors in solution, RAR α homodimers and RAR α–RXR α heterodimers, formed in the absence or the presence of a naturally occurring RA response element (βRARE) under different ionic conditions, were analyzed by size-exclusion fast protein liquid chromatography and sucrose density gradient sedimentation. In the presence of [3H]RA both RAR α and RXR α existed primarily as monomers of 50 kDa in solutions containing 80 mmKCl. However, when βRARE was included in these incubations, a 40-fold increase in the occurrence of both the RAR α homodimers and the RAR α–RXR α heterodimers (125 kDa) was observed. The presence of RAR α and RXR α in the βRARE-associated homo- and heterodimers was confirmed by the positive interaction of the receptors with the specific antibodies. Both RAR α homodimers and RAR α–RXR α heterodimers bound βRARE even in the absence of the ligand RA with the heterodimer showing a 2- to 4-fold greater affinity than the homodimer for the DNA binding element. When the receptors were incubated in solutions of increasing ionic concentration (50–300 mmKCl), a decrease in the amount of both RAR α homodimers and RAR α–RXR α heterodimers was accompanied by a corresponding increase in the monomeric fraction even in the presence of βRARE, suggesting that the high salt concentrations inhibit the surface to surface interactions between the monomers. These observations suggest thatin vivo,as in solution, the formation of a stable retinoid receptor dimer complex is dependent upon both receptor–receptor and receptor–RARE interactions.