Abstract
The members of the NR5A subfamily of nuclear receptors (NRs) are important regulators of pluripotency, lipid and glucose homeostasis, and steroidogenesis. Liver receptor homologue 1 (LRH-1; NR5A2) and steroidogenic factor 1 (SF-1; NR5A1) have therapeutic potential for the treatment of metabolic and neoplastic disease; however, a poor understanding of their ligand regulation has hampered the pursuit of these proteins as pharmaceutical targets. In this study, we dissect how sequence variation among LRH-1 orthologs affects phospholipid (PL) binding and regulation. Both human LRH-1 (hLRH-1) and mouse LRH-1 (mLRH-1) respond to newly discovered medium chain PL agonists to modulate lipid and glucose homeostasis. These PLs activate hLRH-1 by altering receptor dynamics in a newly identified alternate activation function region. Mouse and Drosophila orthologs contain divergent sequences in this region potentially altering PL-driven activation. Structural evidence suggests that these sequence differences in mLRH-1 and Drosophila FTZ-f1 (dmFTZ-f1) confer at least partial ligand independence, making them poor models for hLRH-1 studies; however, the mechanisms of ligand independence remain untested. We show using structural and biochemical methods that the recent evolutionary divergence of the mLRH-1 stabilizes the active conformation in the absence of ligand, yet does not abrogate PL-dependent activation. We also show by mass spectrometry and biochemical assays that FTZ-f1 is incapable of PL binding. This work provides a structural mechanism for the differential tuning of PL sensitivity in NR5A orthologs and supports the use of mice as viable therapeutic models for LRH-1-dependent diseases.
Highlights
NR5A nuclear receptors are important pharmaceutical targets with poorly understood ligand regulation
We have shown that DLPC is able to bind to the Liver receptor homologue 1 (LRH-1) ligand binding domain (LBD) and activate the receptor by altering receptor dynamics at both an alternate activation function surface and the canonical activation function helix (AF-H) [18]
Overall Structure— most the residues contacting bound PLs are conserved in the LRH-1/steroidogenic factor 1 (SF-1) family, residues 419QAGATL424 in human LRH-1 (hLRH-1) are replaced by 438HTEVAF443 in mouse LRH-1 (mLRH-1) as a result of a late evolutionary divergence in the rodent lineage [22]
Summary
NR5A nuclear receptors are important pharmaceutical targets with poorly understood ligand regulation. The human liver receptor homologue-1 (LRH-1;3 NR5A2) is a member of the NR5A class of NRs that regulate the expression of genes central to embryonic development, cell cycle progression, reproduction, and lipid homeostasis [1] in response to activating PLs [2] This family includes steroidogenic factor-1 (SF-1; NR5A1) and Drosophila melanogaster fushi tarazu factor 1 (Ftz-F1; NR5A3). Subsequent mass spectroscopy analysis showed that mLRH-1 is capable of binding to PCs; PL binding was reduced when compared with human NR5A receptors [22] In line with these results, humanization of mLRH-1, by reversing a key sequence substitution in the alternate activation function region, increased sensitivity to PL regulation, suggesting that the mechanism for PL-driven activation has diverged in rodents [22]. We used hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) to show that the mouse-loop sequence stabilizes the alternate activation function surface and AF-H in the absence of PL, whereas only minimally impacting PL binding
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