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.

Figure 1. LXXLL motifs and AF-2 domain are involved in SHP homodimerization

(A) Schematic of FLAG- (F) and myc- (M) SHP constructs used in Co-IP assays. LXXLL and AF-2 residue numbers are in parentheses. Point mutations are in bold and underlined. Black and white lines represent wildtype and mutant LXXLL motifs, respectively. Black and white boxes represent wildtype and mutant AF-2 domain, respectively. (B) Co-IP assay. HEK293 cells were transfected with the indicated combinations of constructs expressing FLAG-SHP (2μg plasmid) and wildtype or mutant myc-SHP (3μg plasmid). Plasmid levels were balanced with empty vector. Whole cell extracts were subjected to IP with FLAG antibody. 25% of the immunoprecipitation reaction was resolved by SDS-PAGE and analyzed by western blotting with FLAG and myc antibodies. Signal was quantitated by laser densitometry. Data for mutants are presented as percent of wildtype protein (100%) bound. Values shown are mean ± SD.

Figure 2. LXXLL motifs and AF-2 domain of SHP are involved in DAX1-SHP heterodimerization

(A) Schematic of FLAG- (F) DAX1 and myc- (M) SHP constructs used in Co-IP assays. LXXLL and AF-2 point mutations are in bold and underlined. Black and white lines represent wildtype and mutant LXXLL motifs, respectively. Black and white boxes represent wildtype and mutant AF-2 domain, respectively. (B and C) SHP LXXLL motif (B) and AF-2 domain (C) involvement in DAX1-SHP heterodimerization. HEK293 cells were transfected with the indicated combinations of FLAG- and myc-epitope tagged expression constructs (2μg each plasmid). Plasmid levels were balanced with empty vector. Whole cell extracts were subjected to IP with FLAG antibody. 25% of the immunoprecipitation reaction was resolved by SDS-PAGE and analyzed by western blotting with FLAG and myc antibodies. Signal was quantitated by laser densitometry. Data for mutants are presented as percent of wildtype protein (100%) bound. Values shown are mean ± SD.

Figure 3. Redundancy of LXXLL motifs in DAX1 homodimerization

(A) Schematic of FLAG- (F) DAX1-N and myc- (M) DAX1 constructs used in Co-IP assays. DAX1-N residue numbers are as in Fig. 3-3. LXXLL point mutations are in bold and underlined, and black and white lines represent wildtype and mutant LXXLL motifs, respectively. (B) Co-IP assay. HEK293 cells were transfected with the indicated combinations of FLAG- and myc-epitope tagged expression constructs (2μg each plasmid). Plasmid levels were balanced with empty vector. Whole cell extracts were subjected to IP with FLAG antibody. 25% of the immunoprecipitation reaction was resolved by SDS-PAGE and analyzed by western blotting with FLAG and myc antibodies. Signal was quantitated by laser densitometry. Data for mutants are presented as percent of wildtype protein (100%) bound. Values shown are mean ± SD.

Figure 4. DAX1A subcellular localization is mostly cytoplasmic, but DAX1-DAX1A heterodimers exist in the nucleus

(A) Nuclear and cytoplasmic fractions of HEK293 cells transfected with combinations of FLAG-DAX1 and myc-DAX1A constructs as indicated (2μg each plasmid) were subjected to western blot analysis with FLAG, myc, and hsp90 antibodies. Plasmid levels were balanced with empty vector. Hsp90 and endogenous c-myc protein were used as cytoplasmic and nuclear markers, respectively. DAX1A localization was observed to be primarily cytoplasmic regardless of the presence of DAX1. (B) Nuclear and cytoplasmic fractions of transfected HEK293 cells were immunoprecipitated with FLAG antibody followed by western blot with FLAG and myc antibodies. Co-IP showed that DAX1-DAX1A heterodimerization occurred mostly in the nucleus. Western analysis in (A) represented 1% Co-IP input.

Figure 5. C-terminus of DAX1 is involved in DAX1-DAX1A heterodimerization

(A) Schematic of FLAG-DAX1 (F) and myc-DAX1A (M) expression constructs. DAX1-N contains residues 1-198, and DAX1-C contains residues 207-470. Note the lack of an AF-2 domain in the CTD. DAX1A residues 390-400 comprise a novel 10 amino acid motif (grey bar). (B) Co-IP assays involved transfection of HEK293 cells with indicated combinations of FLAG- and myc-tagged expression constructs (2μg each plasmid). Plasmid levels were balanced with empty vector. Whole cell extracts were subjected to IP with FLAG antibody followed by western blotting with FLAG and myc antibodies.

Figure 6. DAX1 AF-2 domain is involved in DAX1-DAX1A heterodimerization

(A) Schematic of FLAG- (F) DAX1 and myc- (M) DAX1A constructs used in Co-IP assays. DAX1-C residue numbers are as in Fig. 5. AF-2 residue numbers are in superscript. AF-2 point mutations are in bold and underlined, and black and white boxes represent wildtype and mutant AF-2 domain, respectively. (B) Co-IP assay. HEK293 cells were transfected with the indicated combinations of FLAG- and myc-epitope tagged expression constructs (2μg each plasmid). Plasmid levels were balanced with empty vector. Whole cell extracts were subjected to IP with FLAG antibody. 25% of the immunoprecipitation reaction was resolved by SDS-PAGE and analyzed by western blotting with FLAG and myc antibodies. Signal was quantitated by laser densitometry. Data for mutants are presented as percent of wildtype protein (100%) bound. Values shown are mean ± SD.