Analysis of homo- and heterodimerization of retinoid receptors in solution

To characterize the dimerization of retinoid receptors in solution, RAR alpha homodimers and RAR alpha-RXR alpha heterodimers, formed in the absence or the presence of a naturally occurring RA response element (betaRARE) 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 alpha and RXR alpha existed primarily as monomers of 50 kDa in solutions containing 80 mM KCl. However, when betaRARE was included in these incubations, a 40-fold increase in the occurrence of both the RAR alpha homodimers and the RAR alpha-RXR alpha heterodimers (125 kDa) was observed. The presence of RAR alpha and RXR alpha in the betaRARE-associated homo- and heterodimers was confirmed by the positive interaction of the receptors with the specific antibodies. Both RAR alpha homodimers and RAR alpha-RXR alpha heterodimers bound betaRARE 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 mM KCl), a decrease in the amount of both RAR alpha homodimers and RAR alpha-RXR alpha heterodimers was accompanied by a corresponding increase in the monomeric fraction even in the presence of betaRARE, suggesting that the high salt concentrations inhibit the surface to surface interactions between the monomers. These observations suggest that in vivo, as in solution, the formation of a stable retinoid receptor dimer complex is dependent upon both receptor-receptor and receptor-RARE interactions.