LXXLL motifs and AF-2 domain mediate SHP ( NR0B2) homodimerization and DAX1 ( NR0B1)–DAX1A heterodimerization

Abstract

Small heterodimer partner (SHP; NR0B2) is an unusual orphan member of the nuclear receptor superfamily that functions as a corepressor of other nuclear receptors through heterodimeric interactions. Mutations in SHP are associated with mild obesity and insulin resistance. The protein domain structure of SHP is similar to Dosage-sensitive sex reversal adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1 (DAX1; NR0B1). Mutations in DAX1 cause AHC with associated hypogonadotropic hypogonadism. DAX1A is an alternatively spliced isoform of DAX1 that lacks the last 80 amino acids of the DAX1 C-terminal repressor domain and is replaced by a novel 10-amino acid motif. We have previously shown homodimerization of SHP and DAX1 individually, heterodimerization of DAX1 with SHP, and heterodimerization of DAX1 with DAX1A. In these studies, we investigated the domains and residues of SHP involved in SHP homodimerization and DAX1–SHP heterodimerization and also further characterized DAX1–DAX1 homodimerization and DAX1–DAX1A heterodimerization. We showed involvement of the SHP LXXLL motifs and AF-2 domain in SHP homodimerization and DAX1–SHP heterodimerization. We demonstrated redundancy of the LXXLL motifs in DAX1 homodimerization. While DAX1A subcellular localization is mostly cytoplasmic, DAX1–DAX1A heterodimers existed in the nucleus, suggesting differential functions for DAX1A in each compartment. We showed that the AF-2 domain of DAX1 is involved in DAX1–DAX1A heterodimerization. These results indicate that NR0B family members use similar mechanisms for homodimerization as well as heterodimerization. These resemble coactivator–receptor interactions that may have potential functional consequences for molecular mechanisms of the NR0B family.