NRluc-hER-CRluc

Estrogen receptors (ERs) are ligand-inducible nuclear transcription factors belonging to the superfamily of steroid hormone receptors (1). The endogenous ligands of ERs such as estradiol (E2) (2), an important estrogen in humans, are secreted by endocrine cells and distributed to their targets blood circulation (3). Upon the binding of estrogens to their inactive apoprotein forms in the cytoplasm or nucleus, ERs are transformed into active receptors that can effectively bind to a DNA element (hormone response element, HRE), leading to transcription of genes (3). ERs consist of six functional domains (A–F): the A/B region contains transactivation functions, region C contains DNA-binding domain (DBD), and region E contains ligand-binding domain (LBD) with an activation function (AF-2) (4). The LBD is an antiparallel helical sandwich of three layers formed by twelve α-helices (H1–H12) and one β-turn (5). The helix H12 serves as a lid in the ligand-binding cavity, thus, behaves as a “molecular switch” to prevent or enhance ER from binding to an array of co-activator proteins (6). The binding of ligands induces a reposition of the helices, especially the reorientation of H12, in the LBD, and can generate different functions. For instance, the binding of ER agonists such as E2 or diethylstilbestrol (DES) leads to H12 aligned over the LBD to yield a specific binding site for the consensus LXXLL motif in AF-2; the binding of selective ER modulators (SERMs) such as raloxifene or tamoxifen sterically hinders the H12 positioning to prevent the formation of a binding surface for AF-2 (4). ERs are responsible for the growth, development, and maintenance of the reproductive skeletal, neuronal, and immune systems (6). The deficiency or excess of estrogens can lead to various diseases including osteoporosis and breast carcinoma. A variety of therapeutic strategies have been developed on the basis of the anti-ER mechanism, including the use of SERMs to provide optimal agonist or antagonist activities in ER-expressed tissues. luciferase (Rluc) is a 36-kDa enzyme protein extracted from a bioluminescent soft coral (sea pansy ()) (7). Rluc can catalyze emission of light from substrates; i.e., the oxidation of coelenterazine to coelenteramide generates a green fluorescence (535–550 nm) (8). Rluc can be split into two domains: a large N-terminal domain (amino acids 1–221) and a C-terminal domain (amino acids 230–311) (9). Splitting Rluc into N- and C-terminal fragments destroys its enzymatic activity, resulting in a complete loss of bioluminescence. The enzymatic activity or bioluminescence can be restored if the N- and C-terminal fragments are in close proximity. This led to the development of the split reporter, a novel labeling strategy that has been used in imaging protein–protein interactions (10) and/or protein activity (11) . For instance, protein A is connected with the N-terminal fragment of Rluc, and protein B is connected with the C-terminal fragment of Rluc. Interaction between proteins A and B recovers the enzymatic activity of Rluc by bringing the two fragments of Rluc close together, which allows for recovery of bioluminescence (10). NRluc-hER-CRluc (hERNCRluc) is an optical agent used to image the intramolecular folding of human ER (hER) (6). hERNCRluc consists of four sequentially linked components: an N-terminal Rluc (N-Rluc) fragment, a fragment of hER α-isoform (hERα) containing the LBD (amino acids 281–549, hER), a spacer linker ((GGGGS)), and a C-terminal Rluc (C-Rluc). The included hER fragment (hER) provides a binding cavity for various ligands, and its conformation can be altered upon binding of the ligands. Depending on the produced protein-folding pattern, partial to full complementation of the N-Rluc and C-Rluc occurs, which is measurable with bioluminescence imaging. hERNCRluc is suitable for distinguishing the functionalities in various ER ligands (i.e., agonists, antagonist, or SERMs) with imaging.