Identification of an Endogenous Ligand Bound to a Native Orphan Nuclear Receptor

Abstract

Orphan nuclear receptors have been instrumental in identifying novel signaling pathways and therapeutic targets. However, identification of ligands for these receptors has often been based on random compound screens or other biased approaches. As a result, it remains unclear in many cases if the reported ligands are the true endogenous ligands, – i.e., the ligand that is bound to the receptor in an unperturbed in vivo setting. Technical limitations have limited our ability to identify ligands based on this rigorous definition. The orphan receptor hepatocyte nuclear factor 4 α (HNF4α) is a key regulator of many metabolic pathways and linked to several diseases including diabetes, atherosclerosis, hemophilia and cancer. Here we utilize an affinity isolation/mass-spectrometry (AIMS) approach to demonstrate that HNF4α is selectively occupied by linoleic acid (LA, C18:2ω6) in mammalian cells and in the liver of fed mice. Receptor occupancy is dramatically reduced in the fasted state and in a receptor carrying a mutation derived from patients with Maturity Onset Diabetes of the Young 1 (MODY1). Interestingly, however, ligand occupancy does not appear to have a significant effect on HNF4α transcriptional activity, as evidenced by genome-wide expression profiling in cells derived from human colon. We also use AIMS to show that LA binding is reversible in intact cells, indicating that HNF4α could be a viable drug target. This study establishes a general method to identify true endogenous ligands for nuclear receptors (and other lipid binding proteins), independent of transcriptional function, and to track in vivo receptor occupancy under physiologically relevant conditions.

Identification of the Flavonoid Luteolin as a Repressor of the Transcription Factor Hepatocyte Nuclear Factor 4α

“…More recently, it has been shown that exogenously expressed HNF4␣ in COS-7 cells binds to LA, and this binding is exchangeable. However, LA does not exhibit a significant effect on the HNF4␣ transcriptional activity (8). In the present study, we showed that luteolin is capable of binding to HNF4␣ and that the binding pocket of HNF4␣ occupied by luteolin is far from that occupied by the fatty acid.…”

“…Recently, linoleic acid (LA) was identified as an endogenous ligand for HNF4␣; however, the binding of LA to HNF4␣ does not affect its transcriptional activity (8). More recently, small synthetic molecules, such as BIM5078 and BI6015, were identified as antagonists for HNF4␣.…”

Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor Hepatocyte nuclear factor 4 alpha

“…Another facet of HNF4A biology that remains unresolved is the identity of its endogenous ligand(s). Although historically considered an orphan nuclear receptor, several fatty acids (FA), including linoleic acid, have been identified as ligands for HNF4A (Hertz et al 1998;Palanker et al 2009;Yuan et al 2009). Fatty acids are an attractive class of putative regulators of HNF4A, since the microbiota are known to regulate FA absorption in zebrafish IECs (Semova et al 2012).…”

Suppression of Hepatocyte Proliferation by Hepatocyte Nuclear Factor 4α in Adult Mice

“…Originally designated as an orphan member due to the lack of a known ligand, subsequent studies have identified fatty acids bound in the ligand binding pocket with linoleic acid as the potential endogenous HNF4␣ ligand (5)(6)(7)(8). HNF4␣ is critical for proper hepatic lipid homeostasis, as evidenced by the development of steatosis in Hnf4␣-deficient mouse livers (9).…”