Biology subjects

Pham, U.

Publications and source records attributed to Pham, U..

8 recordsLinked to original sources

bioRxiv2026

BCKDK Inhibitors Directly Antagonize Angiotensin II Type 1 Receptor and Induce Vasodilation Independently of BCAA Catabolism or BCKDK

Branched-chain amino acids (BCAA) have been implicated in diabetes and heart failure, leading to ongoing clinical trials with inhibitors of BCKDK, a key regulator of BCAA catabolism. We have previously shown that these inhibitors promote vasodilation and lower blood pressure, well-known cardioprotective effects, but how this occurs remained unclear. We show here, using explanted vessels, in vivo hemodynamics and telemetry, and genetically modified mice, that the vasodilatory effect of BCKDK inhibitors is direct, rapid, independent of nitric oxide, independent of BCAA catabolism, and most strikingly, independent of BCKDK itself. Instead, we demonstrate, using structural and biochemical studies, that BCKDK inhibitors directly antagonize the angiotensin II type I receptor (AT1R) and consequent angiotensin-mediated vasoconstriction. These data demonstrate a strong off-target effect of BCKDK inhibitors, independent of BCAA or BCKDK, which may explain their cardioprotective effects but also questions if this protection would be additive to existing AT1R blockers.

physiology↗

bioRxiv2025

β-arrestin recruitment facilitates a direct association with G proteins

G protein-coupled receptors (GPCRs) are targets for almost a third of all FDA-approved drugs. GPCRs are known to signal through both heterotrimeric G proteins and {beta}-arrestins. Traditionally these pathways were viewed as largely separable, with G proteins primarily initiating downstream signaling while {beta}-arrestins modulate receptor trafficking and desensitization in addition to regulating their own signaling events. Recent studies suggest an integrated role of G proteins and {beta}-arrestins in GPCR signaling, however the cellular and biochemical requirements for G protein: {beta}-arrestin interactions remain unclear. Here we show that G proteins and {beta}-arrestins can directly interact. Through utilization of {beta}-arrestin-biased receptors and artificially enforced {beta}-arrestin relocalization, we demonstrate that recruitment of {beta}-arrestin to the plasma membrane is sufficient to interact with the G protein Gi. Using purified proteins, we show that Gi directly interacts with {beta}-arrestin. In addition, we find that Gi family members differ in their degree of association with {beta}-arrestin, and that a large degree of this selectivity resides within the alpha helical domain of Gi. These findings delineate the cellular and biochemical conditions that drive direct interactions between G proteins and {beta}-arrestins and illuminate the molecular basis for how they work together to effect GPCR signaling.

biochemistry↗

bioRxiv2024

Location-biased β-arrestin conformations direct GPCR signaling

{beta}-arrestins are multifunctional intracellular proteins that regulate the desensitization, internalization and signaling of over 800 different G protein-coupled receptors (GPCRs) and interact with a diverse array of cellular partners1,2. Beyond the plasma membrane, GPCRs can initiate unique signaling cascades from various subcellular locations, a phenomenon known as "location bias"3,4. Here, we investigate how {beta}-arrestins direct location-biased signaling of the angiotensin II type I receptor (AT1R). Using novel bioluminescence resonance energy transfer (BRET) conformational biosensors and extracellular signal-regulated kinase (ERK) activity reporters, we reveal that in response to the endogenous agonist Angiotensin II and the {beta}-arrestin-biased agonist TRV023, {beta}-arrestin 1 and {beta}-arrestin 2 adopt distinct conformations across different subcellular locations, which are intricately linked to differential ERK activation profiles. We also uncover a population of receptor-free catalytically activated {beta}-arrestins in the plasma membrane that exhibits insensitivity to different agonists and promotes ERK activation on the plasma membrane independent of G proteins. These findings deepen our understanding of GPCR signaling complexity and also highlight the nuanced roles of {beta}-arrestins beyond traditional G protein pathways.

biochemistry↗

bioRxiv2024

ACKR3 Proximity Labeling Identifies Novel G protein- and β-arrestin-independent GPCR Interacting Proteins

The canonical paradigm of GPCR signaling recognizes G proteins and {beta}-arrestins as the two primary transducers that promote GPCR signaling. Recent evidence suggests the atypical chemokine receptor 3 (ACKR3) does not couple to G proteins, and {beta}-arrestins are dispensable for some of its functions. Here, we employed proximity labeling to identify proteins that interact with ACKR3 in cells devoid of {beta}-arrestin. We identified proteins involved in the endocytic machinery and evaluated a subset of proteins conserved across several GPCR-based proximity labeling experiments. We discovered that the bone morphogenic protein 2-inducible kinase (BMP2K) interacts with many different GPCRs with varying dependency on {beta}-arrestin. Together, our work highlights the existence of modulators that can act independently of G proteins and {beta}-arrestins to regulate GPCR signaling and provides important evidence for other targets that may regulate GPCR signaling. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/577545v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@56a61org.highwire.dtl.DTLVardef@18b17baorg.highwire.dtl.DTLVardef@c2bd55org.highwire.dtl.DTLVardef@11c39b0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Subcellular localization of GPCR kinases differentially modulate biased signaling at CXCR3

Some G protein-coupled receptors (GPCRs) demonstrate biased signaling, where ligands of the same receptor differentially activate specific downstream signaling pathways over others. Ligand-specific receptor phosphorylation by GPCR kinases (GRKs) is one mechanism underlying this phenomenon. Recent evidence demonstrates that GPCRs traffic to and signal from subcellular compartments beyond the plasma membrane, a paradigm termed location bias. Here, we show that GRKs translocate to endosomes following stimulation of the chemokine receptor CXCR3 and other GPCRs. The GRK recruitment patterns at the plasma membrane and endosome are distinct and depend on the identity of the ligand used to activate the receptor. Using cells deficient of GRKs, we demonstrate that biased ligands have unique signaling profiles upon rescue of location-specific GRK isoforms. Our work highlights a role of the GRKs in location-biased GPCR signaling and demonstrates the complex interactions between ligand, GRK isoform and cellular location that contribute to biased signaling.

biochemistry↗

bioRxiv2022

Location bias contributes to functionally selective responses of biased CXCR3 agonists

Some G protein-coupled receptor (GPCR) ligands act as "biased agonists" which preferentially activate specific signaling transducers over others. Although GPCRs are primarily found at the plasma membrane, GPCRs can traffic to and signal from many subcellular compartments. Here, we determine that differential subcellular signaling contributes to the biased signaling generated by three endogenous ligands of the chemokine GPCR CXCR3. The signaling profile of CXCR3 changed as it trafficked from the plasma membrane to endosomes in a ligand-specific manner. Endosomal signaling was critical for biased activation of G proteins, {beta}-arrestins, and ERK1/2. In CD8+ T cells, the chemokines promoted unique transcriptional responses predicted to regulate inflammatory pathways. In a mouse model of contact hypersensitivity, {beta}-arrestin-biased CXCR3-mediated inflammation was dependent on receptor internalization. Our work demonstrates that differential subcellular signaling is critical to the overall biased response observed at CXCR3, which has important implications for drugs targeting chemokine receptors and other GPCRs.

biochemistry↗

bioRxiv2021

Revealing A-T and G-C Hoogsteen base pairs in stressed protein-bound duplex DNA

Watson-Crick base pairs (bps) are the fundamental unit of genetic information and the building blocks of the DNA double helix. However, A-T and G-C can also form alternative Hoogsteen bps, expanding the functional complexity of DNA. We developed Hoog-finder, which uses structural fingerprints to rapidly screen Hoogsteen bps, which may have been mismodeled as Watson-Crick in crystal structures of protein-DNA complexes. We uncovered seventeen Hoogsteen bps, seven of which were in complex with six proteins never before shown to bind Hoogsteen bps. The Hoogsteen bps occur near mismatches, nicks, and lesions and some appear to participate in recognition and damage repair. Our results suggest a potentially broad role for Hoogsteen bps in stressed regions of the genome and call for a community-wide effort to identify these bps in current and future crystal structures of DNA and its complexes.

biophysics↗

bioRxiv2020

Hoogsteen base pairs increase the susceptibility of double-stranded DNA to cytotoxic damage

As the Watson-Crick faces of nucleobases are protected in double-stranded DNA (dsDNA), it is commonly assumed that deleterious alkylation damage to the Watson-Crick faces of nucleobases predominantly occurs when DNA becomes single-stranded during replication and transcription. However, damage to the Watson-Crick faces of nucleobases has been reported in dsDNA in vitro through mechanisms that are not understood. In addition, the extent of protection from methylation damage conferred by dsDNA relative to single-stranded DNA (ssDNA) has not been quantified. Watson-Crick base-pairs in dsDNA exist in dynamic equilibrium with Hoogsteen base-pairs that expose the Watson-Crick faces of purine nucleobases to solvent. Whether this can influence the damage susceptibility of dsDNA remains unknown. Using dot-blot and primer extension assays, we measured the susceptibility of adenine-N1 to methylation by dimethyl sulfate (DMS) when in an A-T Watson-Crick versus Hoogsteen conformation. Relative to unpaired adenines in a bulge, Watson-Crick A-T base-pairs in dsDNA only conferred ~130-fold protection against adenine-N1 methylation and this protection was reduced to ~40-fold for A(syn)-T Hoogsteen base-pairs embedded in a DNA-drug complex. Our results indicate that Watson-Crick faces of nucleobases are accessible to alkylating agents in canonical dsDNA and that Hoogsteen base-pairs increase this accessibility. Given the higher abundance of dsDNA relative to ssDNA, these results suggest that dsDNA could be a substantial source of cytotoxic damage. The work establishes DMS probing as a method for characterizing A(syn)-T Hoogsteen base pairs in vitro and also lays the foundation for a sequencing approach to map A(syn)-T Hoogsteen and unpaired adenines genome-wide in vivo.

biochemistry↗