Biology subjects

Barak, L. S.

Publications and source records attributed to Barak, L. S..

7 recordsLinked to original sources

bioRxiv2025

Hidden Bias: GPCR Polymorphisms and the Genomic Landscape of β- Arrestin Signaling

Clinically, no human diseases are currently diagnosed as being directly driven by {beta}- arrestins. Nonetheless, there is growing interest in developing {beta}-arrestin-biased drugs, due to the key regulatory role of {beta}-arrestins in modulating the cellular signaling of most G protein-coupled receptors (GPCRs). In cell-based studies of rhodopsin-family GPCRs, mutations in a conserved proline-hydrophobic (ProH) motif within the second intracellular loop (ICL2) have been shown to alter {beta}-arrestin signaling bias. The clinical relevance of such mutations in humans is unknown. However, if naturally occurring single nucleotide polymorphisms (SNPs) affecting this ProH motif exist across the GPCR family, then cross-referencing these genetic variants with large-scale population sequencing and epidemiologic data could reveal potential roles for {beta}-arrestin signaling in human health. In this report, we identify SNPs in human GPCRs that correspond to ProH substitutions, and we show that, in the neurotensin receptor NTSR1, these variants can indeed shift signaling bias. We also estimate a lower-bound population frequency for such SNPs, suggesting that although rare for any given receptor, their cumulative prevalence across the GPCR superfamily may be large enough to impact phenotypic variation. Together with emerging data from biased ligands, our findings support the idea that genetic variations in {beta}-arrestin signaling could represent a meaningful source of therapeutic relevance. Significance statementMutations altering G protein-coupled receptor (GPCR) signaling can affect health. We have identified a specific amino acid determinant in intracellular loop (ICL2) of human rhodopsin family GPCRs that influences {beta}-arrestin signaling. Although the medical consequences of this determinant remain unclear, we show that single nucleotide polymorphisms (SNPs) affecting the determinant routinely occur. By studying neurotensin receptor NTSR1, we confirm that the SNP alters {beta}-arrestin signaling bias. While individually rare per receptor, these SNPs may collectively contribute to {beta}-arrestin-related phenotypic changes in the human population. Our findings, combined with research on biased drugs, support the idea that {beta}-arrestin signaling might serve as a useful therapeutic target, opening new possibilities for precision medicine.

biochemistry↗

bioRxiv2025

Rare variant in intracellular loop-2 of the ghrelin receptor reveals novel mechanisms of GPCR biased signaling and trafficking

G protein-coupled receptors (GPCRs) are the largest class of integral membrane proteins and the most common pharmaceutical drug target. Through allosteric coupling, GPCRs transduce extracellular stimuli into physiologically-relevant intracellular signaling cascades via G proteins and {beta}-arrestin ({beta}arr). The growth hormone secretagogue receptor (GHSR) is a rhodopsin-like, peptide hormone GPCR considered a promising target for both metabolic and neurological diseases. Here, by characterizing an ultra-rare coding variant in intracellular loop-2 (ICL2) of the GHSR, GHSRL149P (L149P), we establish a unique role of ICL2 in GPCR biased signaling, lipid modulation, and intracellular trafficking. Using an array of bioluminescence resonance energy transfer (BRET)-based assays, we show that the natural L149P mutant exhibits (i) a constitutive plasma membrane [PM] expression bias, (ii) preferential partitioning away from cholesterol-enriched PM microdomains, (iii) enhanced agonist-directed endocytosis, and (iv) dramatic signaling bias towards {beta}arr1/2 over Gq, Gi/o, and G12/13. Using a combination of pharmacological and genetic tools, we demonstrate that {beta}arr1/2 recruitment to L149P requires G protein-coupled receptor kinase-2/3 (GRK2/3)-mediated phosphorylation, but it does not utilize protein kinase C (PKC), G{beta}{gamma}-dependent GRK2/3 translocation, or Gi/o, supporting a G protein-independent mechanism. Lastly, we found that {beta}arr1/2 recruitment to L149P requires both GRK2/3 and GRK5/6, while the wild-type GHSR relies exclusively on GRK2/3, consistent with increased GRK6 pre-coupling to the L149P mutant. Collectively, our findings using a rare, natural variant reveal novel mechanisms of GPCR regulation that could be leveraged to improve personalized medicine and facilitate the rational design/discovery of GPCRICL2-directed drugs. SIGNIFICANCE STATEMENTG protein-coupled receptors (GPCRs) are the largest class of membrane proteins and the most prevalent drug target class in medicine. However, the structural and mechanistic determinants of GPCR signaling and membrane compartmentalization remains incompletely understood. Here, we characterize an ultra-rare natural variant of the growth hormone secretagogue receptor (GHSR), L149P, that uncovers an unanticipated role for intracellular loop 2 (ICL2) in receptor biased signaling, intracellular trafficking, and membrane lipid modulation. The variant exhibits a strong bias for {beta}-arrestin signaling independent of canonical G protein pathways, mediated instead by unique GPCR kinase interactions and a distinct subcellular distribution. These findings reveal previously unrecognized regulatory layers in GPCR structure/function and identify ICL2 as a promising target for designing precision pharmacotherapeutics.

biochemistry↗

bioRxiv2024

Design of allosteric modulators that change GPCR G protein subtype selectivity

G protein-coupled receptors (GPCRs), the largest family of drug targets, can signal through 16 subtypes of G proteins. Biased compounds that selectively activate therapy-relevant pathways promise to be safer, more effective medications. The determinants of bias are poorly understood, however, and rationally-designed, G protein-subtype-selective compounds are lacking. Here, using the prototypical class A GPCR neurotensin receptor 1 (NTSR1), we find that small molecules binding the intracellular GPCR-transducer interface change G protein coupling by subtype-specific and predictable mechanisms, enabling rational drug design. We demonstrate that the compound SBI-553 switches NTSR1 G protein preference by acting both as a molecular bumper and a molecular glue. Structurally, SBI-553 occludes G protein binding determinants on NTSR1, promoting association with select G protein subtypes for which an alternative, shallow-binding conformation is energetically favorable. Minor modifications to the SBI-553 scaffold produce allosteric modulators with distinct G protein subtype selectivity profiles. Selectivity profiles are probe-independent, conserved across species, and translate to differences in in vivo activity. These studies demonstrate that G protein selectivity can be tailored with small changes to a single chemical scaffold targeting the receptor-transducer interface and, as this pocket is broadly conserved, present a strategy for pathway-selective drug discovery applicable to the diverse GPCR superfamily.

pharmacology and toxicology↗

bioRxiv2024

Nonlinear progression across the occult transition establishes cancer lethality

Cancer screening is based upon a linear model of neoplastic growth and malignant progression. Yet, historical observations suggest that malignant progression is uncoupled from growth which may explain the paradoxical increase in early-stage breast cancer detection without a dramatic reduction in metastatic burden. Here we lineage trace millions of genetically transformed field cells and thousands of screen detectable and symptomatic tumors using a cancer rainbow mouse model of HER2+ breast cancer. Transition rates from field cell to screen detectable tumor and then to symptomatic tumors were estimated from a dynamical model of tumor development. Field cells are orders of magnitude less likely to transition to a screen detectable tumor than the subsequent transition of a screen detectable tumor to a symptomatic tumor. Our model supports a critical occult transition in tumor development during which time a transformed cell becomes a bona fide neoplasm. Lineage tracing and test-by-transplantation reveals that nonlinear progression during or prior to the occult transition gives rise to nascent lethal cancers at screen detection. Simulations illustrate how occult transition rates are a critical determinant of tumor growth and malignancy in the lifetime of a host. Our data provides direct experimental evidence that cancers can deviate from the predictable linear progression model foundational to current screening paradigms

cancer biology↗

bioRxiv2024

β-arrestin-biased Allosteric Modulator of Neurotensin Receptor 1 Reduces Ethanol Drinking and Responses to Ethanol Administration in Rodents

Alcohol use disorders (AUDs) impose an enormous societal and financial burden, and world-wide, alcohol misuse is the 7th leading cause of premature death1. Despite this, there are currently only 3 FDA approved pharmacological treatments for the treatment of AUDs in the United States. The neurotensin (Nts) system has long been implicated in modulating behaviors associated with alcohol misuse. Recently, a novel compound, SBI-553, that biases the action of Nts receptor 1 (NTSR1) activation, has shown promise in preclinical models of psychostimulant misuse. Here we investigate the efficacy of this compound to alter ethanol-mediated behaviors in a comprehensive battery of experiments assessing ethanol consumption, behavioral responses to ethanol, sensitivity to ethanol, and ethanol metabolism. Additionally, we investigated behavior in avoidance and cognitive assays to monitor potential side effects of SBI-553. We find that SBI-553 reduces binge-like ethanol consumption in mice without altering avoidance behavior or novel object recognition. We also observe sex-dependent differences in physiological responses to sequential ethanol injections in mice. In rats, we show that SBI-553 attenuates sensitivity to the interoceptive effects of ethanol (using a Pavlovian drug discrimination task). Our data suggest that targeting NTSR1 signaling may be promising to attenuate alcohol misuse, and adds to a body of literature that suggests NTSR1 may be a common downstream target involved in the psychoactive effects of multiple reinforcing substances.

neuroscience↗

bioRxiv2022

Neurotensin receptor allosterism revealed in complex with a biased allosteric modulator

The NTSR1 neurotensin receptor (NTSR1) is a G protein coupled receptor (GPCR) found in the brain and peripheral tissues with neurotensin (NTS) being its endogenous peptide ligand. In the brain, NTS modulates dopamine neuronal activity, induces opioid-independent analgesia, and regulates food intake. Recent studies indicate that biasing NTSR1 toward {beta}-Arrestin signaling can attenuate the actions of psychostimulants and other drugs of abuse. Here we provide the cryoEM structures of NTSR1 ternary complexes with heterotrimeric Gq and Go with and without the brain penetrant small molecule SBI-553. In functional studies, we discovered that SBI-553 displays complex allosteric actions exemplified by negative allosteric modulation for G proteins that are G subunit selective and positive allosteric modulation and agonism for {beta}-Arrestin translocation at NTSR1. Detailed structural analysis of the allosteric binding site illuminated the structural determinants for biased allosteric modulation of SBI-553 on NTSR1. These insights promise to both accelerate the structure-guided design of more effective NTSR1 therapeutics and provide insights into the complexities of GPCR allosteric modulation.

biophysics↗

bioRxiv2020

Establishment of Multi-stage Models of Drug Taking and Seeking in Mice

A genetically tractable animal model would provide a needed strategy to resolve the biological basis of drug addiction. Intravenous self-administration (IVSA) is the gold standard for modeling cocaine and opioid addiction in animals, but technical limitations have precluded the widespread use of IVSA in mice. Here, we describe the first IVSA paradigms for mice that capture the multi-stage nature of the disease and permit predictive modeling. Mice with long-standing indwelling jugular catheters engaged in cocaine or opioid-associated lever responding that was fixed ratio- and dose-dependent, extinguished by the withholding of drug, and reinstated by the presentation of paired cues. Machine learning revealed that vulnerability to drug seeking and relapse were predicted by a mouses a priori response to novelty, sensitivity to drug-induced locomotion, and drug-taking behavior. Application of this behavioral and analysis approach to genetically-engineered mice will facilitate the identification of the neural circuits driving addiction susceptibility and relapse and focused therapeutic development.

neuroscience↗