Heterodimerization with RXR is essential for the high-affinity specific binding of multiple nuclear receptors to their cognate DNA sequences. NR dimerization is a two-step process, initiated in solution by interaction between amino acid residues with helices 9 and 10 of the ligand binding domains of RXR and its NR partners. Studies of the orphan nuclear receptor HNF4 alpha, which forms homodimers exclusively, have indicated that two charged residues in this region, HNF4 alpha(K300) and HNF4 alpha(E327), are key mediators of dimerization. We have analyzed the contribution of the homologous residues in RXR alpha (RXR alpha(E395), RXR alpha(K422)) and PPAR gamma (PPAR gamma(E405), PPAR gamma(K432)) to the formation of the RXR alpha-PPAR gamma heterodimer. Charge reversal mutants of RXR alpha (RXR alpha(E395K), RXR alpha(K422E)) and PPAR gamma (PPAR gamma(E405K,) PPAR gamma(K432E)) show impaired ability to form heterodimers with wild-type PPAR gamma and RXR alpha, respectively. However, pairs of mutants with balanced charge changes, i.e., RXR alpha(E395K) with PPAR alpha(K432E) and RXR alpha(K422E) with PPAR gamma(E405K), are able to form dimers. Ligand response is preserved in the PPAR gamma mutants, indicating the mutation does not result in major structural derangement of the protein. These results establish the importance of salt bridges between these residues in the heterodimerization of nuclear receptors, and offer a technical approach to generating functional NR mutants with directed heterodimerization specificity. Such mutants will be valuable tools in the genetic analysis of NR function. (C) 2007 Elsevier Inc. All rights reserved.