FIG. 3.

Interaction between HNF4α1 and coactivators in vivo and in vitro is inhibited by the presence of the F domain. (A) Interactions between HNF4α1 and coactivators GRIP1 and SRC1a were examined in the yeast two-hybrid assay (Clontech) with various Gal4DBD-HNF4 (pGBT9) and Gal4AD-GRIP1 or -SRC1a (pGAD424) constructs as described in Materials and Methods and previously (10). Shown is one representative experiment of two or more independent transformations into S. cerevisiae SFY526 containing an integrated β-Gal reporter construct. Standard deviations are from four independent clones from a single transformation. Numbers indicate amino acid sequence encoded in the various constructs. NR boxes, nuclear receptor interaction motifs as previously described (10). (B) In vitro pulldown assays between the GST control and GST-GRIP1 (aa 563 to 1121) and in vitro-translated ³⁵S-HNF4α1 constructs as indicated were performed as described in Materials and Methods. Shown is the phosphorimage after SDS-PAGE of eluted material as well as percent binding of input protein (10% input is shown). One of several experiments is shown. Positions of ¹⁴C-labeled MW markers are shown at the left. Negative controls for the pulldown assays shown in panel B, using in vitro-translated ³⁵S-C/EBPα and ³⁵S-luciferase, are not shown. (C) As for panel B. wt, GST-GRIP1 as in panel B; GRIP1 NRmut, as wt GRIP1 except with mutations in NR boxes II and III. The presence of the F domain and AF-2 is indicated for each HNF4α construct.