A Proteomic Microarray Approach for Exploring Ligand-initiated Nuclear Hormone Receptor Pharmacology, Receptor Selectivity, and Heterodimer Functionality

Abstract

Nuclear hormone receptors (NHRs) are major regulators of development and homeostasis in multiple organ systems. These proteins are ligand-modulated transcription factors that regulate gene expression in response to changes in circulating levels of their cognate hormones or hormone analogs. When NHRs bind ligands, they adopt distinct conformations that enable or disable the binding of coregulator proteins in a manner that reflects the agonist versus antagonist character of the ligand. Using the estrogen receptor ligand binding domain as a representative member of the NHR family, we show the development of functional protein microarrays and use them to explore coactivator recruitment and NHR homo-and heterodimer functionality. These NHR protein microarrays can be fabricated in either a forward mode (coactivator recruited to printed NHR) or a reversed mode (NHR recruited to printed coactivator). From these microarrays, we can predict the potency and pharmacological character of various NHR ligands through the nature of their coactivator recruitment. Additionally different coactivator proteins can be functionally classified and their affinity for NHRs can be quantified. NHR-selective antagonist ligands and small molecule coactivator mimics disrupt the coactivator-NHR complex. This novel proteomic approach was also used to assess coactivator recruitment to explore heterodimer functionality. Heterodimers of the estrogen receptor were found only to recruit coactivators when both monomers are bound with agonist ligands, an observation that provides an insight into the complex biology of hormones that act on tissues containing both NHR subtypes. We can extend this NHR proteomic approach to the analysis of multidomain full-length NHR constructs and can concurrently monitor the activation state of different classes of NHRs with a mixture of endogenous or synthetic ligands of varying NHR selectivity and pharmacology. There is great interest in the complex pharmacology of agents that act through members of the nuclear hormone receptor (NHR) 1 superfamily. The human genome project suggests that this superfamily consists of 48 different proteins, most of which are ligand-modulated transcription factors that regulate gene expression in response to changes in circulating levels of their cognate hormones or various hormone analogs (1). Alteration in activity of different NHRs leads to various pathological states from obesity and diabetes mellitus to various hormone-sensitive carcinomas. Although there are some NHRs whose activity is modulated independently of ligand binding (2), the majority of NHRs exist in a physiologically silent state when unliganded. After ligand binding, NHR transcriptional activity is mediated by receptor-ligand interaction with a series of coregulatory proteins, the most well defined of which are the p160 steroid receptor coactivators (SRC1, -2, and -3) (3, 4). The binding of agonists to NHRs stabilizes a conformation that engenders their interaction with the SRCs through the binding of specific nuclear r...

Quantification of Ligand-Regulated Nuclear Receptor Corepressor and Coactivator Binding, Key Interactions Determining Ligand Potency and Efficacy for the Thyroid Hormone Receptor

“…The NdeI-BamHI fragment of the NCoRC' from the GST-NCoRC' vector was excised and cloned into the NdeI and BamHI sites of pET15b vector and expressed as a 6XHis fusion protein. Expression of the NRID fragment of hSRC3 protein (residues 627-829) has been previously described (23). The NRID fragments of SRC3 and NCoR were fluorescently labeled with 5-iodoacetamidofluorescein according to our published procedure (23,24).…”

“…Expression of the NRID fragment of hSRC3 protein (residues 627-829) has been previously described (23). The NRID fragments of SRC3 and NCoR were fluorescently labeled with 5-iodoacetamidofluorescein according to our published procedure (23,24). …”

Exploration of Dimensions of Estrogen Potency

“…Unlike previous studies of ER-coactivator interactions, which used a synthetic peptide containing a single LXXLL motif as the coactivator component (21)(22)(23), our use of a complete NRID fragment of SRC3 in the present study resulted in binding curves displaying clear saturation for all of the estrogens and revealed considerable variations in affinities. The ER␣ and ER␤ LBDs and SRC3 NRID peptide fragments used in the present study have been extensively characterized in a number of our previous studies that demonstrated that these fragments displayed functional characteristics very similar to full-length ERs in terms of ligand binding and ligand-specific conformational and receptor dimerization changes as well as agonist/antagonist-regulated coactivator recruitment profiles (15,17,18,(27)(28)(29). The subnanomolar sensitivity with which our tr-FRET assay measures SRC3-ER interaction, together with nearly stoichiometric binding of SRC3 to ER at equilibrium with an EC 50 of 0.88 nM for ER␣ and 0.76 nM for ER␤ in the coactivator titration assay, supports the fact that the ER LBDs and SRC3 NRID fragments that we have used retain most if not all of the receptor-coactivator interaction properties of full-length ER and SRC3 proteins.…”

Large-Scale Protein-Protein Interaction Detection Approaches: Past, Present and Future

“…In general, the weakness of cell-free protein expression approaches is the lack of post-translational modifi cations and appropriate folding of some proteins (7). However, it was suggested that addition of chaperonin GroEL can alleviate those problems (45).…”

“…In addition, a high-throughput method of fl uorescence labeling of full-length cDNA products has been reported (45). The labeling relies on fl ourophore-tagged puromycin in conjunction with either a release factor-free expression system or exclusion of stop codons.…”