Nuclear receptors (NRs) are ligand-dependent transcription factors that regulate the expression of genes involved in virtually all aspects of physiology and disease 
. The identification of ligand-receptor pairs began with the pioneering work of Jensen 
, Edelman 
and others who injected radioactive ligands into animals and observed their binding to nuclear receptor proteins. These experiments had an inherent bias in that the probe ligand displaced binding of the true endogenous ligand, which could not be detected in these assays. The validity of the classical steroid receptor-ligand pairs are now well established, but the limitations of existing approaches leave open the possibility that additional ligands may exist for the classical steroid receptors. Indeed, such speculation was raised for estrogen receptor some time ago, as well as more recently 
, and for intestinal vitamin D receptor that is activated by an enterohepatic bile acid 
The assignment of endogenous ligands to the so-called orphan nuclear receptors is even more equivocal 
. Typically, orphan receptors are screened in transcription-based assays against random compound collections that include natural or synthetic molecules; in other cases candidate ligands are identified based on their structure or biological activity 
. Alternatively, compounds are added at supra-pharmacologic doses and their metabolic products are found to be ligands 
. Most recently, ligands have been reported based on fortuitous binding to heterologously produced recombinant proteins 
. Such studies have been of enormous value and have lead to the identification of many novel signaling pathways, drugs and therapeutic targets. Nonetheless, ligands have been identified for only about half of the 48 human nuclear receptors and the tally in non-human species is even lower. Furthermore, it remains unclear how many of the ligands that have been identified are the actual ligands that are bound to the receptor in vivo
. For example, the first orphan receptor-ligand pair to be identified was 9-cis retinoic acid and RXR 
, yet it appears unlikely that sufficient 9-cis retinoic acid exists in vivo
to serve as a true endogenous ligand 
, NR2A1) is another orphan receptor whose endogenous ligand remains unclear 
. HNF4α is essential to early development and plays critical roles in hepatocyte differentiation 
and in homeostasis of the adult liver, intestine, and pancreatic beta cells 
. In humans, mutations in the coding and promoter regions of HNF4α lead to Maturity Onset Diabetes of the Young 1 (MODY1), a heritable form of type 2 diabetes 
. Recent crystallographic studies identified a mixture of tightly bound fatty acids in the ligand binding pocket (LBP) of bacterially expressed HNF4α and HNF4γ 
, but it remains unclear what ligands are bound when the receptor is expressed in its native physiological environment. There have also been somewhat controversial reports of fatty acyl Co-enzyme A thioesters as HNF4α ligands 
These studies highlight the need to distinguish between ligands that may bind under non-native conditions and those that are the true endogenous ligands. The most rigorous definition of a true endogenous ligand is a compound that binds the LBP in vivo in the absence of experimental probes or other perturbations. Identification of ligands by this definition requires new technical approaches.
In addition to identification of endogenous ligands for orphan (and other) nuclear receptors, new experimental tools to identify ligands are also needed to address the role of ligands in the evolution of the nuclear receptor superfamily 
. Examination of HNF4 is also instructive in this regard as it is present in the earliest metazoan organisms and is one of the most evolutionarily conserved nuclear receptors 
. Therefore, the question of whether HNF4 binds an endogenous ligand, the identity of that ligand and its effect on HNF4 transcriptional activity is of particular interest and may be relevant to the entire receptor superfamily. However, these issues cannot be addressed without an assay that can identify potential ligands in the absence of a pre-supposed function.
Here we use an affinity isolation/mass spectrometry (AIMS) approach to identify the endogenous ligand that is bound to HNF4α in mammalian cells and in mouse liver. The approach is unbiased in that it does not make any pre-assumptions as to what the ligand might be or how it might affect HNF4α function. Our results indicate that the vast majority of HNF4α is bound to a single essential fatty acid: linoleic acid (LA). Furthermore, our results show that the binding is reversible, indicating that LA is an exchangeable ligand. To our knowledge, this represents the first time an endogenous ligand has been identified by virtue of its association with a nuclear receptor in animal tissue.