Biology subjects

Namkung, Y.

Publications and source records attributed to Namkung, Y..

5 recordsLinked to original sources

bioRxiv2026

Allosteric pathways govern Gα protein coupling selectivity at promiscuous GPCRs

G protein-coupled receptors (GPCRs) regulate diverse physiological responses by engaging distinct heterotrimeric G proteins, yet the basis of G selectivity in promiscuous receptors remains unclear. Although ligand bias holds therapeutic promise, selectivity has been assumed to reside mainly in the ligand-binding site (LBS) or G protein interface (GPI). Here, we combine whole-receptor mutagenesis, functional G protein assays, molecular dynamics simulations, interpretable machine learning method, and Bayesian network modeling to identify residue networks governing Gq/11 and G12/13 coupling and to define the molecular basis of G protein preference and promiscuity at two vasopressor GPCRs, the angiotensin II type 1 and prostaglandin F2 receptors. While residues within the LBS and GPI domains contribute to coupling efficiency and subtype discrimination, we find that long-range allosteric communication across the receptor, including from structurally unresolved domains, is the principal determinant of G protein preference and promiscuity. These allosteric pathways integrate multiple receptor domains, confer signaling robustness to mutation, and hierarchically govern coupling preferences. Our findings suggest that G protein selectivity is an allosterically encoded property of GPCRs and provide a conceptual framework for designing ligands and receptors with tailored G protein-biased signaling.

pharmacology and toxicology↗

bioRxiv2026

Interpretable Machine Learning Model of Receptor Dynamics Reveals AT1R Allostery and a Negative Allosteric Modulator

Allosteric modulation of G protein-coupled receptors (GPCRs) offers major advantages in receptor selectivity and signaling control; yet systematic approaches to identify allosteric modulators, define their binding sites, and map the underlying allosteric networks remain limited. Current molecular dynamics (MD) and machine learning (ML)-based methods often rely on correlation-driven or black-box models that provide limited mechanistic insight. We developed an interpretable probabilistic framework that extracts residue-level dependencies from MD ensembles using Bayesian network modeling (BNM). By representing each residue through its local interaction energy, BNM identifies both local and long-range energetic couplings and maps the allosteric communication pathways linking the AngII binding site to the G-protein interface in the angiotensin II type 1 receptor (AT1R). To functionally prioritize these pathways, we integrated BNM with comprehensive mutational analysis, combining whole-receptor alanine mutagenesis data with exhaustive in silico deep mutational scanning to validate BNM-predicted hotspots. This approach recovered state-dependent allosteric communities, revealed residues in noncanonical regions that regulate Gq coupling and identified positions whose functional importance emerged only with specific, predicted substitutions, as well as highlighted a cryptic intracellular pocket enriched in communication hubs. Guided by these network-derived residues and pocket geometries, structure-based virtual screening identified a small, fragment-like molecule negative allosteric modulator (NAM) named Q2 that attenuates AngII-mediated Gq signaling. Mutational mapping supports Q2 binding adjacent to the G-protein interface, consistent with its mechanism of action. Together, these results establish a generalizable and interpretable framework for uncovering GPCR allosteric communication networks and discovering modulators that exploit these networks.

molecular biology↗

bioRxiv2025

Tunable Bias Signaling of the Angiotensin II Type 1 Receptor for Inotropy via C-Terminal Peptide Modifications and Allosteric Site Targeting

The angiotensin II (AngII) type 1 receptor (AT1R) is a key prototypical G protein-coupled receptor in cardiovascular regulation. Biased agonists that activate G protein or {beta}-arrestin pathways provide promising therapeutic potential, but the molecular determinants for this signaling bias and its physiological implications remain poorly understood. This study profiles AngII analogs with modifications at the C-terminal Phe8, revealing that analogs 11, 12, and 29a exhibit varying degrees of Gq engagement while maintaining potent {beta}-arrestin recruitment. Notably, 12 enhances left ventricular ejection fraction with minimal pressor responses in normotensive rats, while other analogs with variable Gq activity do not promote inotropy. Molecular modeling indicates that the unique profile of 12 results from its flexible long side chain engaging a deep allosteric pocket within AT1R. This study demonstrates that engineering AngIIs C-terminus enables selective tuning of AT1R signaling to control arterial versus cardiac responses, providing strategies for developing improved cardiovascular therapeutics. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/670122v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@52b49corg.highwire.dtl.DTLVardef@1cf92dcorg.highwire.dtl.DTLVardef@b2c103org.highwire.dtl.DTLVardef@19db799_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Development of a genetically encoded melanocortin sensor for high sensitivity intravital imaging

The central melanocortin system, composed of peptides derived from pro-opiomelanocortin (POMC) such as the melanocyte-stimulating hormones (-, {beta}-, {gamma}-MSH) and melanocortin 4 receptors (MC4R), along with the agouti-related protein (AgRP), plays a pivotal role in controlling energy balance. To elucidate the dynamic role of -MSH release in regulating appetite, specific, sensitive, and spatiotemporally resolved genetic sensors are required. The melanocortin 1 receptor (MC1R) scaffold was leveraged for its robust plasma membrane expression, high affinity for melanocortin peptides and low affinity for AgRP to design a -MSH selective sensor for in vivo use. This was achieved by integrating circularly permuted green fluorescent protein (cpGFP) into the receptor, which we named Fluorescence Amplified Receptor sensor for Melanocortin (FLAREMC). The FLAREMC sensor shows high potency and selectivity in heterologous and homologous expressing cells for -MSH and the synthetic melanocortin agonist MTII but not to the inverse agonist AgRP. The sensor exhibited impaired signaling, with reduced G protein activation, no {beta}-arrestin coupling, and failed to internalize upon agonist stimulation. In vitro, FLAREMC displayed high photostability and reversible photoactivation. These properties suggest that the FLAREMC is suitable for long-term activity recording in the brain without desensitizing or interfering with endogenous melanocortin receptor signaling. When expressed in the paraventricular nucleus (PVN) of the mouse hypothalamus, the primary site of anorexigenic -MSH signaling, FLAREMC demonstrated its effectiveness in detecting changes associated with melanocortin responses in vivo. FLAREMC enables the study of melanocortin system in cultured cells and intravitally. This first of its class highly sensitive melanocortin sensor will serve as a valuable tool to advance our understanding of the complex dynamics governing melanocortin-dependent appetite regulation and related processes in the brain.

neuroscience↗

bioRxiv2022

Allosteric modulation of GPCR-induced β-arrestin trafficking and signaling by a synthetic intrabody

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of {beta}-arrestin ({beta}arr) recruitment and trafficking. For several GPCRs, such as the vasopressin type II receptor (V2R), which exhibit high affinity for {beta}arrs, agonist-stimulation first drives the translocation of {beta}arrs to the plasma membrane, followed by endosomal trafficking. We previously found that mutation of a single phosphorylation site in V2R (i.e., V2RT360A) results in near-complete loss of {beta}arr translocation to endosomes although {beta}arrs are robustly recruited to the plasma membrane. Here, we show that a synthetic intrabody referred to as intrabody30 (Ib30), which selectively recognizes an active-like {beta}arr1 conformation, rescues endosomal translocation of {beta}arr1 for V2RT360A. In addition, Ib30 also rescues agonist-induced ERK1/2 MAP kinase activation for V2RT360A to levels similar to that of the wild-type V2R. Molecular dynamics simulations reveal that Ib30 binding promotes active-like conformation in {beta}arr1 with respect to the inter-domain rotation. Interestingly, we also observe that Ib30 enhances the interaction of {beta}arr1 with {beta}2-adaptin, which provides a mechanistic basis for the ability of Ib30 to promote endosomal trafficking of {beta}arr1. Taken together, our data provide a novel mechanism to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can potentially be integrated in the current paradigm of GPCR-targeted drug discovery. SignificanceThe interaction of G protein-coupled receptors (GPCRs) with {beta}-arrestins ({beta}arrs) is a critical step in their regulatory and signaling paradigms. While intrabodies that bind to GPCRs, G proteins and {beta}arrs have been utilized as biosensors and regulators of functional outcomes, allosteric targeting of receptor-transducer complexes to encode gain of function has not been documented so far. Here, we discover that a conformation-specific synthetic intrabody recognizing GPCR-bound {beta}arr1 can allosterically enhance endosomal trafficking of {beta}arr1 and agonist-induced ERK1/2 MAP kinase activation. This intrabody promotes an active-like {beta}arr1 conformation and enhances the interaction of {beta}2-adaptin with {beta}arr1. Our findings establish a conceptual framework to allosterically modulate protein-protein interactions in GPCR signaling cascade to modulate their trafficking and signaling responses.

biochemistry↗