Nuclear hormone receptors (NRs) are ligand-dependent transcription factors that regulate target gene transcription. We report the molecular cloning and characterization of a novel human cDNA encoding TRAM-1, a thyroid hormone receptor activator molecule, a ϳ160-kDa protein homologous with SRC-1/TIF2, by far-Western-based expression screening. TRAM-1 binds to thyroid hormone receptor (TR) and other NRs in a liganddependent manner and enhances ligand-induced transcriptional activity of TR. The AF-2 region in NRs has been thought to play a critical role in mediating ligand-dependent transactivation by the interaction with coactivators. Surprisingly, TRAM-1 retains strong ligand-dependent interaction with an AF-2 mutant of TR (E457A), while SRC-1 fails to interact with this mutant. Furthermore, we identified a critical TRAM-1 binding site in rat TR␤1 outside of AF-2, as TRAM-1 shows weak ligand-dependent interaction with a helix 3 ligand binding domain TR mutant (K288A), compared with SRC-1. These results suggest that TRAM-1 is a coactivator that may exhibit its activity by interacting with subdomains of NRs other than the AF-2 region, in contrast to SRC-1/TIF2.The nuclear hormone receptors (NRs) 1 regulate target gene transcription in response to various ligands including steroids, thyroid hormone (T 3 ), retinoids, and vitamin D. The liganded NRs bind to their cognate response elements, located in the promoter regions of target genes, and stimulate transcription by the interaction with coactivators. Functional analysis of NRs has shown that there are two major activation domains.The N-terminal domain (AF-1) contains a ligand-independent activation function, whereas the extreme C-terminal region of the ligand binding domain (AF-2) exhibits ligand-dependent transactivation (1). The AF-2 region is conserved among NRs, and deletion or point mutations in this region impair transcriptional activation without changing ligand and DNA binding affinities (2-4). Recent x-ray crystallographic studies of the ligand binding domain of NRs revealed that the ligand induces a major conformational change in the AF-2 region (5-7), suggesting that this region may play a critical role in mediating transactivation by a ligand-dependent interaction with coactivators. Several putative coactivators of NRs have been identified, using either far-Western or yeast two-hybrid techniques, including SRC-1, RIP140, TIF1, TIF2, and CREB binding protein (CBP)/p300 (8 -14). As expected, these proteins fail to interact with AF-2 mutants of .Using bacterially expressed thyroid hormone receptor (TR) as a probe, a cDNA expression library was screened, and a novel cDNA encoding TRAM-1, a putative thyroid hormone receptor activator molecule, was isolated. TRAM-1 belongs to a nuclear receptor coactivator (NCoA) gene family that includes SRC-1 and TIF2. TRAM-1 exhibits ligand-dependent, and unexpectedly, AF-2-independent interaction with TR, a biochemical feature distinct from SRC-1/TIF2. Our findings suggest that TRAM-1 may serve a novel pathway for transcr...

Tirzepatide (LY3298176) is a dual GIP and GLP-1 receptor agonist under development for the treatment of type 2 diabetes mellitus (T2DM), obesity, and nonalcoholic steatohepatitis. Early phase trials in T2DM indicate that tirzepatide improves clinical outcomes beyond those achieved by a selective GLP-1 receptor agonist. Therefore, we hypothesized that the integrated potency and signaling properties of tirzepatide provide a unique pharmacological profile tailored for improving broad metabolic control. Here, we establish methodology for calculating occupancy of each receptor for clinically efficacious doses of the drug. This analysis reveals a greater degree of engagement of tirzepatide for the GIP receptor than the GLP-1 receptor, corroborating an imbalanced mechanism of action. Pharmacologically, signaling studies demonstrate that tirzepatide mimics the actions of native GIP at the GIP receptor but shows bias at the GLP-1 receptor to favor cAMP generation over β-arrestin recruitment, coincident with a weaker ability to drive GLP-1 receptor internalization compared with GLP-1. Experiments in primary islets reveal β-arrestin1 limits the insulin response to GLP-1, but not GIP or tirzepatide, suggesting that the biased agonism of tirzepatide enhances insulin secretion. Imbalance toward GIP receptor, combined with distinct signaling properties at the GLP-1 receptor, together may account for the promising efficacy of this investigational agent.

Nuclear hormone receptors activate gene transcription through ligand-dependent association with coactivators. Specific LXXLL sequence motifs present in these cofactors are sufficient to mediate these ligand-induced interactions. A thyroid hormone receptor (TR)-binding protein (TRBP) was cloned by a Sos-Ras yeast twohybrid system using TR␤1-ligand binding domain as bait. TRBP contains 2063 amino acid residues, associates with TR through a LXXLL motif, and is ubiquitously expressed in a variety of tissues and cells. TRBP strongly transactivates through TR␤1 and estrogen receptor in a dose-related and ligand-dependent manner, and also exhibits coactivation through AP-1, CRE, and NFB-response elements, similar to the general coactivator CBP͞p300. The C terminus of TRBP binds to CBP͞p300 and DRIP130, a component of the DRIP͞TRAP͞ARC complex, which suggests that TRBP may activate transcription by means of such interactions. Further, the association of TRBP with the DNA-dependent protein kinase (DNA-PK) complex and DNA-independent phosphorylation of TRBP C terminus by DNA-PK point to a potential connection between transcriptional control and chromatin architecture regulation. H ormone-regulated gene activation mediated by nuclear receptors relies on high-affinity interactions between their ligand-binding domains (LBDs) and transcriptional coactivators. Upon ligand induction, coactivators harboring LXXLL motifs are recruited to the target gene promoter as a preformed complex (1-3). The coactivator complex includes CBP͞p300 (4-6), pCAF (7, 8), steroid receptor coactivator 1 (SRC-1) family members (9, 10), an RNA coactivator SRA (11), and an arginine transmethylase CARM1 (12). A biochemically purified DRIP͞TRAP͞ARC complex containing mediator͞srb subunits is also recruited by nuclear receptors in a ligand-dependent manner (13-16). The histone acetylase-containing coactivator and vitamin D receptor-interacting protein (DRIP) complexes then act in concert to remodel chromatin and to regulate gene activation. We report here the identification and characterization of a ubiquitous LXXLL-containing protein, thyroid hormone receptor (TR)-binding protein (TRBP), that acts as a general coactivator. The association of TRBP with a DRIP component and CBP͞p300, as well as the DNA-dependent protein kinase (DNA-PK) complex, suggests an important link between the coactivator complex and DNA-PK function (17). Materials and MethodsYeast Two-Hybrid Screen and TRBP cDNA Cloning. The Sos-Ras yeast two-hybrid system was used to screen a rat pituitary cDNA library, as previously described (18), except that screen was performed in the presence of 20 M 3,3Ј,5-triiodothyroacetic acid (Triac; Sigma), a hydrophilic 3,3Ј,5-triiodo-L-thyronine (T 3 ) analog. Rat TR␤1-LBD (292-461) was PCR amplified and inserted in-frame with the C terminus of human Sos as bait. The cDNA sequence derived from the screen was used to search the GenBank database with the BLAST program. The partial 3Ј cDNA clone of human TRBP (KIAA0181, anonymous sequence) identified in th...