Phosphorylation of Mineralocorticoid Receptor Ligand Binding Domain Impairs Receptor Activation and Has a Dominant Negative Effect over Non-phosphorylated Receptors

Abstract

Post-translational modification of steroid receptors allows fine-tuning different properties of this family of proteins, including stability, activation, or interaction with co-regulators. Recently, a novel effect of phosphorylation on steroid receptor biology was described. Phosphorylation of human mineralocorticoid receptor (MR) on Ser-843, a residue placed on the ligand binding domain, lowers affinity for agonists, producing inhibition of gene transactivation. We now show that MR inhibition by phosphorylation occurs even at high agonist concentration, suggesting that phosphorylation may also impair coupling between ligand binding and receptor activation. Our results demonstrate that agonists are able to induce partial nuclear translocation of MR but fail to produce transactivation due at least in part to impaired co-activator recruitment. The inhibitory effect of phosphorylation on MR acts in a dominant-negative manner, effectively amplifying its functional effect on gene transactivation.Steroid receptors (SRs) 3 are part of the nuclear receptor superfamily of ligand-dependent transcription factors that modulate gene transcription in response to changes in steroid hormone levels (1). SRs present a modular architecture, with three well defined domains: an NH 2 -terminal activation domain (NTD), a central DNA binding domain, and a COOHterminal ligand binding domain (LBD). Generally, the apo receptor resides in the cytosol forming a heterocomplex with other proteins including chaperones such as heat-shock protein 90 (Hsp90). Ligand binding alters SR conformation, releasing it from the complex and promoting translocation to the nucleus, where it binds specific DNA sequences as a dimer and alters transcription of target genes by recruiting transcriptional co-regulators (2). Post-translational modifications, including phosphorylation, ubiquitylation, sumoylation, and acetylation, modulate SR stability and different steps on the activation pathway, including receptor dimerization, DNA binding, and interaction with co-regulators (3).Until recently, it was widely assumed that SR ligand affinity is an intrinsic property defined by the structure of the LBD and not subject to modulation by post-translational modifications. However, Shibata et al. (4) have shown that regulated phosphorylation of Ser-843 in the LBD of human mineralocorticoid receptor (MR), a canonical member of the SR subfamily of nuclear receptors, impairs its ability to mediate gene transactivation by lowering ligand affinity. MR is closely related to the glucocorticoid receptor (GR) and can be activated under physiological conditions by mineralocorticoids such as aldosterone and glucocorticoids such as cortisol or corticosterone. MR has a wide variety of physiological and pathophysiological functions, with a prominent role in regulating transepithelial ion and fluid transport, which is essential for extracellular volume homeostasis and, therefore, blood pressure control. Phosphorylation of MR Ser-843 is restricted to intercalated cells of the distal ...

30 YEARS OF THE MINERALOCORTICOID RECEPTOR: Evolution of the mineralocorticoid receptor: sequence, structure and function

“…Figure 5 also shows the α-helices on the MR as determined by X-ray crystallography (Bledsoe et al 2005, Fagart et al 2005, Edman et al 2015 and amino acids that have been found to be important in either steroid binding or transcriptional activation of the MR or CR (Fagart et al 1998, Geller et al 2000, Bledsoe et al 2005, Hultman et al 2005, Bridgham et al 2006, Shibata et al 2013, Edman et al 2015, Jimenez-Canino et al 2016, Mani et al 2016 or the GR (Bledsoe et al 2002, He et al 2014, Edman et al 2015, Mani et al 2016. A striking feature, discussed in more detail below, is the strong conservation of amino acids among the CR, MR, GR, PR and AR, including lamprey PR and CR.…”

“…However, because intercalated cells lack 11β-HSD2, activation of the intercalated cell MR by de-phosphorylation is followed by F or B acting as an MR agonist (Funder 2013, Shibata et al 2013. Interestingly, high potassium levels increase phosphorylation of Ser-843 (Funder 2013, Shibata et al 2013, Jimenez-Canino et al 2016. Phosphorylated Ser-843 human MR has only been found in intercalated cells in the kidney distal tubule; other tissues such as brain, aorta, heart and colon do not contain phosphorylated Ser-843.…”

“…In a second pathway, progesterone is hydroxylated at C17 and then hydroxylated at C21 to form 11-deoxycortisol, which is hydroxylated at C11 to form cortisol (Baker 2011, Rossier et al 2015. Fagart et al 2005, Edman et al 2015 and GR (Bledsoe et al 2002, He et al 2014 and functional studies of human MR mutants (Fagart et al 1998, Geller et al 2000, Bledsoe et al 2005, Shibata et al 2013, Jimenez-Canino et al 2016, Mani 2016 to investigate the evolution of selectivity for Aldo and other corticosteroids by the MR in terrestrial vertebrates (Baker et al 2013) and ray-finned fish (Prunet et al 2006, Bury & Sturm 2007, Arterbery et al 2011, Sugimoto et al 2016, as well as the basis for binding of some glucocorticoids by human MR and other vertebrate MRs , Mani et al 2016. Together, these data provide molecular and structural fingerprints for investigating the evolution of selectivity for 3-ketosteroids by the MR in vertebrates.…”

Evolution of the Mineralocorticoid Receptor: Sequence, Structure and Function

“…To gain a deeper understanding of the evolution of the MR, we have taken advantage of the sequencing, in the last five years, of a cornucopia of genomes from vertebrates at key evolutionary transitions, including lamprey, elephant shark and coelacanth, to investigate regions of conservation and divergence among and between MRs and GRs. We use these sequence analyses, the crystal structures human MR (Bledsoe, et al 2005;Edman, et al 2015;Fagart, et al 2005;) and GR (Bledsoe, et al 2002;He, et al 2014) and functional studies of human MR mutants (Bledsoe et al 2005;Fagart, et al 1998;Geller et al 2000;Jimenez-Canino, et al 2016;Li et al 2005;Shibata, et al 2013,Mani, 2016 to investigate the evolution of selectivity for Aldo and other corticosteroids by the MR in terrestrial vertebrates (Baker et al 2013) and ray-finned fish (Arterbery, et al 2011;Bury and Sturm 2007;Prunet, et al 2006;Sugimoto et al 2016), as well as the basis for binding of some glucocorticoids by human MR and other vertebrate MRs Mani et al 2016). Together these data provide molecular and structural fingerprints for investigating the evolution of selectivity for 3ketosteroids by the MR in vertebrates.…”

“…Interestingly, high potassium levels increase phosphorylation of Ser-843 (Funder 2013;Jimenez-Canino et al 2016;Shibata et al 2013 phosphorylated in vivo, then the evolution of a corresponding proline in the GR lobe-finned and ray-finned fishes would provide a mechanism for specificity for regulation of transcriptional activation of the MR through a kinase/phosphatase that would not affect the GR.…”