The Orphan Nuclear Receptor SHP Utilizes Conserved LXXLL-Related Motifs for Interactions with Ligand-Activated Estrogen Receptors

SHP (short heterodimer partner) is an unusual orphan nuclear receptor consisting only of a ligand-binding domain, and it exhibits unique features of interaction with conventional nuclear receptors. While the mechanistic basis of these interactions has remained enigmatic, SHP has been suggested to inhibit nuclear receptor activation by at least three alternatives; inhibition of DNA binding via dimerization, direct antagonism of coactivator function via competition, and possibly transrepression via recruitment of putative corepressors. We now show that SHP binds directly to estrogen receptors via LXXLL-related motifs. Similar motifs, referred to as NR (nuclear receptor) boxes, are usually critical for the binding of coactivators to the ligand-regulated activation domain AF-2 within nuclear receptors. In concordance with the NR box dependency, SHP requires the intact AF-2 domain of agonist-bound estrogen receptors for interaction. Mutations within the ligand-binding domain helix 12, or binding of antagonistic ligands, which are known to result in an incomplete AF-2 surface, abolish interactions with SHP. Supporting the idea that SHP directly antagonizes receptor activation via AF-2 binding, we demonstrate that SHP variants, carrying either interaction-defective NR box mutations or a deletion of the repressor domain, have lost the capacity to inhibit agonist-dependent transcriptional estrogen receptor activation. Furthermore, our studies indicate that SHP may function as a cofactor via the formation of ternary complexes with dimeric receptors on DNA. These novel insights provide a mechanistic explanation for the inhibitory role of SHP in nuclear receptor signaling, and they may explain how SHP functions as a negative coregulator or corepressor for ligand-activated receptors, a novel and unique function for an orphan nuclear receptor.

SHP（short heterodimer partner，异二聚体短伴侣）是一类特殊的孤儿核受体（orphan nuclear receptor），仅由配体结合域（ligand-binding domain）构成，且具备与经典核受体（conventional nuclear receptors）相互作用的独特性质。尽管这类相互作用的机制基础长期以来始终成谜，但已有研究提示SHP可通过至少三种途径抑制核受体的激活：经由二聚化作用抑制DNA结合、通过竞争直接拮抗共激活因子（coactivator）的功能，以及可能通过招募潜在共抑制因子（corepressors）实现转录抑制。本研究证实，SHP可通过LXXLL相关基序（LXXLL-related motifs）直接结合雌激素受体（estrogen receptors）。此类被称为NR（核受体，nuclear receptor）框的基序，通常是共激活因子结合核受体配体调控激活结构域AF-2（AF-2 domain）的关键位点。契合NR框的依赖特性，SHP与激动剂结合型雌激素受体的相互作用，需要受体具备完整的AF-2结构域。已知配体结合域螺旋12的突变，或是拮抗剂配体（antagonistic ligands）的结合，均可导致AF-2表面结构不完整，进而阻断与SHP的相互作用。为验证SHP可通过结合AF-2直接拮抗受体激活这一假说，我们发现携带相互作用缺陷型NR框突变或缺失阻遏结构域的SHP变异体，已丧失抑制激动剂依赖型转录雌激素受体激活的能力。此外，本研究还表明，SHP可通过在DNA上与二聚化受体形成三元复合物（ternary complexes）的方式发挥共因子功能。这些全新的研究发现为SHP在核受体信号通路中的抑制作用提供了机制层面的合理解释，同时也阐明了SHP如何作为负向共调节因子或共抑制因子调控配体激活型受体——这是孤儿核受体所具备的一种新颖且独特的生理功能。