Biology subjects

Irannejad, R.

Publications and source records attributed to Irannejad, R..

8 recordsLinked to original sources

bioRxiv2023

Selective activation of intracellular β1AR using a spatially restricted antagonist

G-protein-coupled receptors (GPCRs) regulate several physiological and pathological processes and represent the target of approximately 30% of FDA-approved drugs. GPCR-mediated signaling was thought to occur exclusively at the plasma membrane. However, recent studies have unveiled their presence and function at subcellular membrane compartments. There is a growing interest in studying compartmentalized signaling of GPCRs. This requires development of novel tools to separate GPCRs signaling at the plasma membrane from the ones initiated at intracellular compartments. We took advantage of the structural and pharmacological information available for {beta}1-adrenergic receptor ({beta}1AR), an exemplary GPCR that functions at subcellular compartments, and rationally designed spatially restricted antagonists. We generated a cell impermeable {beta}1AR antagonist by conjugating a suitable pharmacophore to a sulfonate-containing fluorophore. This cell-impermeable antagonist only inhibited {beta}1AR on the plasma membrane. In contrast, a cell permeable {beta}1AR agonist containing a non-sulfonated fluorophore, efficiently inhibited both the plasma membrane and Golgi pools of {beta}1ARs. Furthermore, the cell impermeable antagonist selectively inhibited the phosphorylation of downstream effectors of PKA proximal to the plasma membrane in adult cardiomyocytes while {beta}1AR intracellular pool remained active. Our tools offer promising avenues for investigating compartmentalized {beta}1AR signaling in various context, potentially advancing our understanding of {beta}1AR-mediated cellular responses in health and disease. They also offer a general strategy to study compartmentalized signaling for other GPCRs in various biological systems.

cell biology↗

bioRxiv2023

Anionic phospholipids determine G protein coupling selectivity at distinct subcellular membrane compartments

G protein-coupled receptors (GPCRs) activate different signaling pathways through coupling to heterotrimeric G proteins consisting of four types of G alpha (G) subunits and the beta gamma (yy) subunits. The C-terminal regions of G proteins is thought to determine coupling selectivity. However, there are several reports indicating that some receptors are promiscuous and can couple to multiple G proteins. The precise mechanism promoting promiscuous coupling is not fully understood. Here we show that anionic phospholipids such as phosphoinositide 4,5 bisphosphate (PI(4,5)P2) promote electrostatic interactions between anionic lipid head groups and positively charged amino acids in the transmembrane 4 (TM4) region of beta adrenergic receptors ({beta}ARs) to prime receptor coupling to the cognate Gs and non-cognate Gi proteins. The promiscuous coupling can only occur at the plasma membrane, a membrane compartment enriched in PI(4,5)P2, while receptors only couple to cognate Gs proteins in compartments that are enriched in less negatively charged phosphoinositides such PI4P at the Golgi membranes. We took advantage of the rapamycin dimerization system to rapidly and inducibly deplete PI(4,5)P2 by recruiting a phosphatase to the plasma membrane and found that in the absence of anionic phospholipids, {beta}ARs couple only to their cognate Gs protein. Finally, we found that mutating {beta}ARs PI(4,5)P2 binding motifs or depleting PI(4,5)P2 results in enhanced {beta}ARs-mediated cAMP response. Together, these findings reveal a role for anionic phosphoinositides in regulating GPCR activity at different subcellular compartments.

cell biology↗

bioRxiv2023

Inhibition of muscarinic receptor signaling protects human enteric inhibitory neurons against platin chemotherapy toxicity

GI toxicity is a common dose-limiting adverse effect of platin chemotherapy treatment. Up to 50% of cancer survivors continue to experience symptoms of chronic constipation or diarrhea induced by their chemotherapy for many years after their treatment. This drug toxicity is largely attributed to damage to enteric neurons that innervate the GI tract and control GI motility. The mechanisms responsible for platin-induced enteric neurotoxicity and potential preventative strategies have remained unknown. Here, we use human pluripotent stem cell derived enteric neurons to establish a new model system capable of uncovering the mechanism of platin-induced enteric neuropathy. Utilizing this scalable system, we performed a high throughput screen and identified drug candidates and pathways involved in the disease. Our analyses revealed that excitotoxicity through muscarinic cholinergic signaling is a key driver of platin-induced enteric neuropathy. Using single nuclei transcriptomics and functional assays, we discovered that this disease mechanism leads to increased susceptibility of specific neuronal subtypes, including inhibitory nitrergic neurons, to platins. Histological assessment of the enteric nervous system in platin-treated patients confirmed the selective loss of nitrergic neurons. Finally, we demonstrated that pharmacological and genetic inhibition of muscarinic cholinergic signaling is sufficient to rescue enteric neurons from platin excitotoxicity in vitro and can prevent platin-induced constipation and degeneration of nitrergic neurons in mice. These studies define the mechanisms of platin-induced enteric neuropathy and serve as a framework for uncovering cell type-specific manifestations of cellular stress underlying numerous intractable peripheral neuropathies.

pharmacology and toxicology↗

bioRxiv2022

GPCR-dependent spatiotemporal cAMP generation confers functional specificity in cardiomyocytes and cardiac responses

Cells interpret a variety of signals through G protein-coupled receptors (GPCRs) and stimulate the generation of second messengers such as cyclic adenosine monophosphate (cAMP). A long-standing puzzle is deciphering how GPCRs elicit different physiological responses despite generating similar levels of cAMP. We previously showed that some GPCRs generate cAMP from both the plasma membrane and the Golgi apparatus. Here, we demonstrate that cardiomyocytes distinguish between subcellular cAMP inputs to elicit different physiological outputs. We show that generating cAMP from the Golgi leads to regulation of a specific PKA target that increases the rate of cardiomyocyte relaxation. In contrast, cAMP generation from the plasma membrane activates a different PKA target that increases contractile force. We further validated the physiological consequences of these observations in intact zebrafish and mice. Thus, we demonstrate that the same GPCR acting through the same second messenger regulates cardiac contraction and relaxation dependent on its subcellular location.

cell biology↗

bioRxiv2020

Regulation of Dopamine D1 Receptor Signaling at the Golgi Apparatus by a Low Affinity Dopamine Transporter

Dopamine is the main catecholamine in the brain and the kidney, where it controls a number of physiological functions such as locomotion, cognition, emotion, endocrine regulation and renal function. As a membrane impermeant hormone and neurotransmitter, dopamine is thought to signal by binding and activating dopamine receptors, members of the G protein couple receptor (GPCR) family, only on the plasma membrane. Here, using novel nanobody-based biosensors, we demonstrate for the first time that the dopamine D1 receptor (D1DR), the primary mediator of dopaminergic signaling in the brain and kidney, not only functions on the plasma membrane but becomes activated at the Golgi apparatus in the presence of its ligand. We present evidence that activation of the Golgi pool of D1DR is dependent on Organic Cation Transporter 2 (OCT2), a dopamine transporter, providing an explanation for how the membrane impermeant dopamine accesses subcellular pools of D1DR. We further demonstrate that dopamine activates Golgi-D1DR in the striatal medium spiny neurons (MSN) and this activity depends on OCT2 function. We also introduce a new approach to selectively interrogate compartmentalized D1DR signaling by inhibiting Gs coupling, using a nanobody-based chemical recruitment system. Using this strategy, we show that Golgi-localized D1DRs regulate cAMP production and mediate local protein kinase A activation. Together, our data suggest that spatially compartmentalized signaling hubs are previously unappreciated regulatory aspects of D1DR signaling. Our data also provide further evidence for the role of transporters in regulating subcellular GPCR activity.

cell biology↗

bioRxiv2020

G protein-regulated endocytic trafficking of adenylyl cyclase type 9

GPCRs are increasingly recognized to initiate signaling via heterotrimeric G proteins as they move through the endocytic network, but little is known about how relevant G protein effectors are localized. Here we report dynamic trafficking of adenylyl cyclase type 9 (AC9) from the plasma membrane to endosomes, while adenylyl cyclase type 1 (AC1) remains in the plasma membrane, and stimulation of AC9 trafficking by ligand-induced activation of Gs-coupled GPCRs or Gs. AC9 transits a similar dynamin-dependent early endocytic pathway as activated GPCRs but, in contrast to GPCR trafficking which is regulated by {beta}-arrestin but not Gs, AC9 trafficking is regulated by Gs but not {beta}-arrestin. We also show that AC9, but not AC1, contributes to cAMP production from endosomes. These results reveal dynamic and isoform-specific trafficking of adenylyl cyclase in the endocytic network, and a discrete role of a heterotrimeric G protein in controlling subcellular location of a relevant effector.

cell biology↗

bioRxiv2019

Unraveling the functional role of the orphan solute carrier, SLC22A24 in the transport of steroid conjugates through metabolomic and genome-wide association studies

Variation in sex hormone levels has wide implications for health and disease. The genes encoding the proteins involved in steroid disposition represent key determinants of interindividual variation in steroid levels and ultimately, their effects. Beginning with metabolomic data from genome-wide association studies (GWAS), we observed that genetic variants in the orphan transporter, SLC22A24 were significantly associated with levels of androsterone glucuronide and etiocholanolone glucuronide (sentinel SNPs p-value <1x10-30). In cells over-expressing human or various mammalian orthologs of SLC22A24, we showed that steroid conjugates and bile acids were substrates of the transporter. Phylogenetic, genomic, and transcriptomic analyses suggested that SLC22A24 has a specialized role in the kidney and appears to function in the reabsorption of organic anions, and in particular, anionic steroids. Phenome-wide analysis showed that functional variants of SLC22A24 are associated with human disease such as cardiovascular diseases and acne, which have been linked to dysregulated steroid metabolism. Collectively, these functional genomic studies reveal a previously uncharacterized protein involved in steroid homeostasis, opening up new possibilities for SLC22A24 as a pharmacological target for regulating steroid levels.\n\nAuthor SummarySteroid hormones, ranging from sex steroids such as testosterone to glucocorticoids play key roles in human health and disease. Accordingly, the identification of the genes and proteins involved in their synthesis, disposition and elimination has been the subject of numerous genetic studies. We have been intrigued by recent studies demonstrating that genetic variants in or near a gene encoding SLC22A24 are strongly associated with steroid levels. SLC22A24 is an orphan transporter with no known ligands and no known biological functions. In this study, we use cellular and computational methods to show that SLC22A24 transports steroid conjugates, bile acids and other dicarboxylic acids. Based on the direction of association of a common stop codon in SLC22A24 with lower levels of steroids, our studies suggest that the transporter functions to reabsorb steroid conjugates in the kidney, a surprising finding, given that conjugation pathways generally function to polar molecules that are readily eliminated by the kidney. The absence of the transporter gene in many species and its presence in higher order primates suggest that SLC22A24 plays a specialized role in steroid homeostasis. Overall, our studies indicate that SLC22A24 functions in the reabsorption of conjugated steroids in the kidney.

pharmacology and toxicology↗

bioRxiv2019

Golgi localized β1-adrenergic receptors stimulate Golgi PI4P hydrolysis by PLCε to regulate cardiac hypertrophy

Increased adrenergic tone resulting from cardiovascular stress leads to development of heart failure, in part, through chronic stimulation of {beta}1 adrenergic receptors ({beta}ARs) on cardiac myocytes. Blocking these receptors is part of the basis for {beta}-blocker therapy for heart failure. Recent data demonstrate that G protein-coupled receptors (GPCRs), including {beta}ARs, are activated intracellularly, although the biological significance is unclear. Here we investigated the functional role of Golgi {beta}ARs in cardiac myocytes and found they activate Golgi localized, prohypertrophic, phosphoinositide hydrolysis, that is not accessed by cell surface {beta}AR stimulation. This pathway is accessed by the physiological neurotransmitter norepinephrine (NE) via an Oct3 organic cation transporter. Blockade of Oct3 or specific blockade of Golgi resident {beta}1ARs prevents NE dependent cardiac myocyte hypertrophy. This clearly defines a physiological pathway activated by internal GPCRs in a biologically relevant cell type and has implications for development of more efficacious {beta}-blocker therapies.

cell biology↗