Figure 4.

PGC-1α physically interacts with FXR in vitro and in cells. (A) Schematic representation of the domain structures of FXRα3 and the summary of the results to determine an interaction between PGC-1α (1-400) and various fragments of FXRα3. The domain structures of FXRα3 are shown (top). Interactions between PGC-1α (1-400) and the indicated fragments of FXRα3 are represented by + or - (right). (B) Mapping of the 5′ interaction domain of FXR with PGC-1α (1-400). ³⁵S-labeled proteins corresponding to each of the four full-length FXR isoforms or 5′ deletions of FXRα3 or FXRα4 were incubated with GST-PGC-1α (1-400) in the presence of glutathione beads as described in Materials and Methods. The bound proteins were separated by SDS-PAGE and detected by autoradiography. (C) Mapping of the 3′ interaction domain of FXR with PGC-1α (1-400). The indicated N-terminal fragments of FXRα3 were in vitro labeled with ³⁵S-methionine and used to determine the interaction with GST-PGC-1α (1-400), as described in B. (D) The DBD of FXR interacts directly with PGC-1α. ³⁵S-labeled full-length PGC-1α protein was used to determine the interaction with the DBD or DBD plus the four amino acid insert (MYTG) of FXR. (E) Mapping the interaction domain of PGC-1α with FXR. 3′ deletions of PGC-1α with or without the mutation of the LXXLL motif were in vitro labeled with ³⁵S-methionine and used to test interaction with the DBD of FXR. (F) FXR ligand has no effect on the interaction between FXR and PGC-1α. ³⁵S-labeled, full-length FXRα1-4 were incubated with PGC-1α (1-400) in the presence or absence of GW4064 (1 μM). The assay was performed as described in B. (G) PGC-1α interacts with FXR in cells. CV-1 cells were transfected with CMX-Flag-PGC-1α and CMX-FXR plasmids, as indicated. After 48 h, whole-cell lysates were prepared and incubated with anti-Flag antibody. The immunoprecipitates were analyzed by SDS-PAGE/Western blot, using an anti-FXR antibody.