Biology subjects

Smrcka, A. V.

Publications and source records attributed to Smrcka, A. V..

6 recordsLinked to original sources

bioRxiv2024

Functional interrogation of cellular Lp(a) uptake by genome-scale CRISPR screening

An elevated level of lipoprotein(a), or Lp(a), in the bloodstream has been causally linked to the development of atherosclerotic cardiovascular disease and calcific aortic valve stenosis. Steady state levels of circulating lipoproteins are modulated by their rate of clearance, but the identity of the Lp(a) uptake receptor(s) has been controversial. In this study, we performed a genome-scale CRISPR screen to functionally interrogate all potential Lp(a) uptake regulators in HuH7 cells. Strikingly, the top positive and negative regulators of Lp(a) uptake in our screen were LDLR and MYLIP, encoding the LDL receptor and its ubiquitin ligase IDOL, respectively. We also found a significant correlation for other genes with established roles in LDLR regulation. No other gene products, including those previously proposed as Lp(a) receptors, exhibited a significant effect on Lp(a) uptake in our screen. We validated the functional influence of LDLR expression on HuH7 Lp(a) uptake, confirmed in vitro binding between the LDLR extracellular domain and purified Lp(a), and detected an association between loss-of-function LDLR variants and increased circulating Lp(a) levels in the UK Biobank cohort. Together, our findings support a central role for the LDL receptor in mediating Lp(a) uptake by hepatocytes.

genetics↗

bioRxiv2023

Internalized beta2-Adrenergic Receptors Inhibit Subcellular Phospholipase C-Dependent Cardiac Hypertrophic Signaling

Chronically elevated neurohumoral drive, and particularly elevated adrenergic tone leading to {beta}-adrenergic receptor ({beta}-AR) overstimulation in cardiac myocytes, is a key mechanism involved in the progression of heart failure. {beta}1-AR and {beta}2-ARs are the two major subtypes of {beta}-ARs present in the human heart, however, they elicit different or even opposite effects on cardiac function and hypertrophy. For example, chronic activation of {beta}1ARs drives detrimental cardiac remodeling while {beta}2AR signaling is protective. The underlying molecular mechanisms for cardiac protection through {beta}2ARs remain unclear. Here we show that {beta}2-AR protects against hypertrophy through inhibition of PLC{varepsilon} signaling at the Golgi apparatus. The mechanism for {beta}2AR-mediated PLC inhibition requires internalization of {beta}2AR, activation of Gi and G{beta}{gamma} subunit signaling at endosomes and ERK activation. This pathway inhibits both angiotensin II and Golgi-{beta}1-AR-mediated stimulation of phosphoinositide hydrolysis at the Golgi apparatus ultimately resulting in decreased PKD and HDAC5 phosphorylation and protection against cardiac hypertrophy. This reveals a mechanism for {beta}2-AR antagonism of the PLC{varepsilon} pathway that may contribute to the known protective effects of {beta}2-AR signaling on the development of heart failure.

pharmacology and toxicology↗

bioRxiv2021

Identification of G Protein αi Signaling Partners by Proximity Labeling Reveals a Network of Interactions that Includes PDZ-RhoGEF

G protein-coupled receptors (GPCRs) that couple to the Gi family of G proteins are key regulators of cell and tissue physiology. Our recent work has discovered novel roles for Gi in migration of neutrophils and fibrosarcoma cells downstream of activated chemoattractant receptors, but the molecular target(s) of Gi in these processes remain to be identified. We adopted an intact cell proximity-based labeling approach using BioID2 coupled to tandem mass tag (TMT)-based quantitative proteomics to identify proteins that selectively interact with the GTP-bound form of Gi1. Multiple targets were identified and validated for selective biotinylation by active BioID2-Gi1(Q204L), suggesting a previously unappreciated network of interactions for activated Gi proteins in intact cells. Extensive characterization of one candidate protein, PDZ-RhoGEF (PRG), revealed that active-Gi1 strongly activates PRG. Strikingly, large differences in the ability of Gi1, Gi2, and Gi3 isoforms to activate PRG were observed despite over 85% sequence identity. We also demonstrate the functional relevance of the interaction between active Gi and PRG ex vivo in primary human neutrophils. Identification and characterization of new targets regulated by Gi both individually and in networks provide insights that will aid not only in investigation of diverse functional roles of Gi-coupled GPCRs in biology but also in the development of novel therapeutic approaches. SummaryProximity-based labeling approach was used to identify signaling networks and signaling mechanisms downstream of Gi-coupled receptors.

pharmacology and toxicology↗

bioRxiv2020

A Universal Allosteric Mechanism for G Protein Activation

G proteins play a central role in signal transduction and pharmacology. Signaling is initiated by cell-surface receptors, which promote GTP binding and the dissociation of G from the G{beta}{gamma} subunits. Structural studies have revealed the molecular basis for subunit association with receptors, RGS proteins and downstream effectors. In contrast, the mechanism of subunit dissociation is poorly understood. We used cell signaling assays, MD simulations, biochemistry and structural analysis to identify a conserved network of amino acids that dictates subunit release. In the presence of the terminal phosphate of GTP, a glycine forms a polar network with an arginine and glutamate, putting torsional strain on the subunit binding interface. This "G-R-E motif" secures GTP and, through an allosteric link, discharges the G{beta}{gamma} dimer. Replacement of network residues prevents subunit dissociation, regardless of agonist or GTP binding. These findings reveal the molecular basis for the final committed step of G protein activation. HIGHLIGHTSO_LIReceptors promote GTP-GDP exchange and dissociation of G protein and {beta}{gamma} subunits C_LIO_LIWe find an allosteric network linking the {gamma} phosphate of GTP with release of G{beta}{gamma} C_LIO_LIThe network consists of a conserved Gly-Arg-Glu "activation triad" C_LIO_LITriad mutations prevent subunit dissociation, regardless of agonist or GTP binding C_LIO_LITriad mutations are responsible for human endocrine and neurological disorders C_LI

biochemistry↗

bioRxiv2020

β-arrestin mediates communication between plasma membrane and intracellular GPCRs to regulate signaling

It has become increasingly apparent that G protein-coupled receptor (GPCR) localization is a master regulator of cell signaling. However, the molecular mechanisms involved in this process are not well understood. To date, observations of intracellular GPCR activation can be organized into two categories: a dependence on OCT3 cationic channel-permeable ligands or the necessity of endocytic trafficking. Using CXC chemokine receptor 4 (CXCR4) as a model, we identified a third mechanism of intracellular GPCR signaling. We show that independent of membrane permeable ligands and endocytosis, upon stimulation, plasma membrane and internal pools of CXCR4 are post-translationally modified and collectively regulate EGR1 transcription. We found that {beta}-arrestin-1 (arrestin 2) is necessary to mediate communication between plasma membrane and internal pools of CXCR4. Notably, these observations may explain that while CXCR4 overexpression is highly correlated with cancer metastasis and mortality, plasma membrane localization is not. Together these data support a model were a small initial pool of plasma membrane-localized GPCRs are capable of activating internal receptor-dependent signaling events.

cell biology↗

bioRxiv2019

Activation of phospholipase C β by Gβγ and Gαq involves C-terminal rearrangement to release auto-inhibition

Phospholipase C (PLC) enzymes hydrolyse phosphoinositide lipids to inositol phosphates and diacylglycerol. Direct activation of PLC{beta} by Gq and/or G{beta}{gamma} subunits mediates signalling by Gq and some Gi coupled G protein-coupled receptors (GPCRs), respectively. PLC{beta} isoforms contain a unique C-terminal extension, consisting of proximal and distal C-terminal domains (CTD) separated by a flexible linker. The structure of PLC{beta}3 bound to Gq is known, however, for both Gq and G{beta}{gamma}, the mechanism for PLC{beta} activation on membranes is unknown. We examined PLC{beta}2 dynamics on membranes using hydrogen deuterium exchange mass spectrometry (HDX-MS). G{beta}{gamma} caused a robust increase in dynamics of the distal C-terminal domain (CTD). Gq showed decreased deuterium incorporation at the Gq binding site on PLC{beta}. In vitro G{beta}{gamma}-dependent activation of PLC is inhibited by the distal CTD. The results suggest that disruption of auto-inhibitory interactions with the CTD, respectively, leads to increased PLC{beta} hydrolase activity.

biochemistry↗