Manipulation of reciprocal salt bridges at the heterodimerization interface alters the dimerization properties of mouse RXRalpha and PPARgamma1.
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 HNF4alpha, which forms homodimers exclusively, have indicated that two charged residues in this region, HNF4alpha(K300) and HNF4alpha(E327), are key mediators of dimerization. We have analyzed the contribution of the homologous residues in RXRalpha (RXRalpha(E395), RXRalpha(K422)) and PPARgamma (PPARgamma(E405), PPARgamma(K432)) to the formation of the RXRalpha-PPARgamma heterodimer. Charge reversal mutants of RXRalpha (RXRalpha(E395K), RXRalpha(K422E)) and PPARgamma (PPARgamma(E405K), PPARgamma(K432E)) show impaired ability to form heterodimers with wild-type PPARgamma and RXRalpha, respectively. However, pairs of mutants with balanced charge changes, i.e., RXRalpha(E395K) with PPARgamma(K432E) and RXRalpha(K422E) with PPARgamma(E405K), are able to form dimers. Ligand response is preserved in the PPARgamma 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.
Chan
Lap Shu
LS
Molecular and Cellular Biology, Sunnybrook Research Institute, 2075 Bayview Avenue, T2-058 Toronto, Ont., Canada M4N 2M5.
Wells
Richard A
RA
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D003198:Computer Simulation; D019281:Dimerization; D051379:Mice; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D047495:PPAR gamma / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D047490:Retinoid X Receptor alpha / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012492:Salts / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
D047495:PPAR gamma; D047490:Retinoid X Receptor alpha; D012492:Salts