Participation of Critical Residues from the Extreme C-Terminal End of the Human Androgen Receptor in the Ligand Binding Function

Abstract

A short C-terminal end is present at the end of the human androgen receptor (hAR) similar to that of other steroid receptors. It is located directly after helix 12 of the ligand binding domain and has never been described as being part of the hydrophobic binding pocket. Although some fragmentary data have indicated the involvement of this region in ligand binding, its precise function still remains unclear. To gain deeper insight into the role of the hAR extreme C-terminal end, an extensive mutational analysis was carried out by using site-directed mutagenesis and alanine scanning over the 13-residue C-terminal end region. Both ligand binding and transcriptional activity were tested with each mutant. Our study demonstrates the participation of almost all of the amino acids in this region for the ligand binding function and consequently for the transcriptional activity. A conformational study by limited proteolysis was performed with the mutants that most affected the affinity of the receptor. It was remarkable that the mutants with a low binding affinity adopted an inactive conformation and were either less or not able to undergo a following conformational change to provide the active form of the receptor. Our results demonstrate the importance of hydrophobicity for the function of the C-terminal end with residues located at very precise positions. Especially, both hydrophobicity and aromaticity on position 916 are critical for providing the correct ligand binding conformation of the receptor. Furthermore, this study highlights essential criteria regarding the C-terminal amino acids which could be applied to other steroid receptors.

Role of Regulatory F-domain in Hepatocyte Nuclear Factor-4α Ligand Specificity

“…The net effect of these differential influences of the F-domain on HNF-4␣ affinity for LCFA-CoA versus LCFA was that deletion of the C-terminal F-domain completely reversed the ligand specificity of the HNF-4␣. Consistent with this finding, almost all of the F-domain of the androgen receptor (AR) participates in ligand binding (46). Similarly, deletion of the C-terminal F-domain in the PR greatly reduces affinity for or abrogates binding of progesterone but minimally affects affinity for antagonist RU486 (reviewed in Ref.…”

“…Also, mutations in the 4th from last residue of the glucocorticoid receptor and mineralocorticoid receptor significantly decrease binding of both agonists and antagonists (reviewed in Ref. 46). In contrast, deletion of the C-terminal F-domain in the estrogen receptor increases affinity for estradiol by 11-fold but oppositely affects the activity of 4-hydroxytamoxifen, a partial agonist/antagonist (47).…”

“…The high flexibility and inability to assign structure to the C-terminal F-domain residues is also evident in the closely related RXR and retinoic acid receptor (revised in Ref. 46). By modulating the structure of the ligand binding E-domain, the F-domain may also control the binding of coactivators/correpressors, possibly due to conformationally induced alterations upon association of putative ligands at its binding pocket (12).…”

A novel mutation (c.T3816 > C) in the androgen receptor gene in a 46,XY female patient with androgen insensitivity syndrome

“…The correct structure is needed to convert an inactive form to an active one of the receptor upon binding of androgens. It is also involved in receptor dimerisation and transcriptional regulation [8,9].…”

Androgen Insensitivity Syndrome (AIS)

“…54 The cluster of mutations between residue 827 and 870 map to subdomains distant from the ligand-binding pocket such as activation function 2, a regulatory surface cleft termed binding function 3, and a region that tethers the C-terminal tail of the receptor. 55,56 The most common C-terminal mutation reported in the androgen receptor (H917R) is associated with a phenotype of complete androgen insensitivity syndrome. 57,58 The DNA-binding domain comprises 70 aminoacids and is highly conserved within the nuclear receptor family.…”