Kavisha Arora

Multi-drug Resistance Protein 4 (MRP4)-mediated Regulation of Fibroblast Cell Migration Reflects a Dichotomous Role of Intracellular Cyclic Nucleotides

Background: MRP4 is an endogenous transporter of cyclic nucleotides that can regulate cell migration. The role of MRP4 in fibroblast migration is unknown. Results: MRP4-deficient fibroblasts migrate faster and have a moderately higher level of intracellular cyclic nucleotides. Conclusion: Inhibition of MRP4 increases fibroblast migration via alteration of intracellular cyclic nucleotide levels. Significance: Inhibition of MRP4 facilitates wound repair. It has long been known that cyclic nucleotides and cyclic nucleotide-dependent signaling molecules control cell migration. However, the concept that it is not just the absence or presence of cyclic nucleotides, but a highly coordinated balance between these molecules that regulates cell migration, is new and revolutionary. In this study, we used multidrug resistance protein 4 (MRP4)-expressing cell lines and MRP4 knock-out mice as model systems and wound healing assays as the experimental system to explore this unique and emerging concept. MRP4, a member of a large family of ATP binding cassette transporter proteins, localizes to the plasma membrane and functions as a nucleotide efflux transporter and thus plays a role in the regulation of intracellular cyclic nucleotide levels. Here, we demonstrate that mouse embryonic fibroblasts (MEFs) isolated from Mrp4−/− mice have higher intracellular cyclic nucleotide levels and migrate faster compared with MEFs from Mrp4+/+ mice. Using FRET-based cAMP and cGMP sensors, we show that inhibition of MRP4 with MK571 increases both cAMP and cGMP levels, which results in increased migration. In contrast to these moderate increases in cAMP and cGMP levels seen in the absence of MRP4, a robust increase in cAMP levels was observed following treatment with forskolin and isobutylmethylxanthine, which decreases fibroblast migration. In response to externally added cell-permeant cyclic nucleotides (cpt-cAMP and cpt-cGMP), MEF migration appears to be biphasic. Altogether, our studies provide the first experimental evidence supporting the novel concept that balance between cyclic nucleotides is critical for cell migration.

Compartmentalization of cyclic nucleotide signaling: a question of when, where, and why?

Preciseness of cellular behavior depends upon how an extracellular cue mobilizes a correct orchestra of cellular messengers and effector proteins spatially and temporally. This concept, termed compartmentalization of cellular signaling, is now known to form the molecular basis of many aspects of cellular behavior in health and disease. The cyclic nucleotides cyclic adenosine monophosphate and cyclic guanosine monophosphate are ubiquitous cellular messengers that can be compartmentalized in three ways: first, by their physical containment; second, by formation of multiple protein signaling complexes; and third, by their selective depletion. Compartmentalized cyclic nucleotide signaling is a very prevalent response among all cell types. In order to understand how it becomes relevant to cellular behavior, it is important to know how it is executed in cells to regulate physiological responses and, also, how its execution or dysregulation can lead to a pathophysiological condition, which forms the scope of the presented review.

Compartmentalized Accumulation of cAMP near Complexes of Multidrug Resistance Protein 4 (MRP4) and Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Contributes to Drug-induced Diarrhea

Background: Diarrhea is an adverse side effect associated with many therapeutics. Results: Irinotecan induced hyperactive cystic fibrosis transmembrane conductance regulator (CFTR) function by inhibiting multidrug resistance protein 4 (MRP4) and formation of MRP4-CFTR macromolecular complexes. Conclusion: MRP4-CFTR-containing macromolecular complexes play an important role in drug-induced diarrhea. Significance: These studies help define molecular mechanisms of drug-induced diarrhea. Diarrhea is one of the most common adverse side effects observed in ∼7% of individuals consuming Food and Drug Administration (FDA)-approved drugs. The mechanism of how these drugs alter fluid secretion in the gut and induce diarrhea is not clearly understood. Several drugs are either substrates or inhibitors of multidrug resistance protein 4 (MRP4), such as the anti-colon cancer drug irinotecan and an anti-retroviral used to treat HIV infection, 3′-azido-3′-deoxythymidine (AZT). These drugs activate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated fluid secretion by inhibiting MRP4-mediated cAMP efflux. Binding of drugs to MRP4 augments the formation of MRP4-CFTR-containing macromolecular complexes that is mediated via scaffolding protein PDZK1. Importantly, HIV patients on AZT treatment demonstrate augmented MRP4-CFTR complex formation in the colon, which defines a novel paradigm of drug-induced diarrhea.

Stabilizing rescued surface-localized δf508 CFTR by potentiation of its interaction with Na(+)/H(+) exchanger regulatory factor 1.

Cystic fibrosis (CF) is a recessive genetic disease caused by mutations in CFTR, a plasma-membrane-localized anion channel. The most common mutation in CFTR, deletion of phenylalanine at residue 508 (ΔF508), causes misfolding of CFTR resulting in little or no protein at the plasma membrane. The CFTR corrector VX-809 shows promise for treating CF patients homozygous for ΔF508. Here, we demonstrate the significance of protein–protein interactions in enhancing the stability of the ΔF508 CFTR mutant channel protein at the plasma membrane. We determined that VX-809 prolongs the stability of ΔF508 CFTR at the plasma membrane. Using competition-based assays, we demonstrated that ΔF508 CFTR interacts poorly with Na+/H+ exchanger regulatory factor 1 (NHERF1) compared to wild-type CFTR, and VX-809 significantly increased this binding affinity. We conclude that stabilized CFTR–NHERF1 interaction is a determinant of the functional efficiency of rescued ΔF508 CFTR. Our results demonstrate the importance of macromolecular-complex formation in stabilizing rescued mutant CFTR at the plasma membrane and suggest this to be foundational for the development of a new generation of effective CFTR-corrector-based therapeutics.

Drug-induced secretory diarrhea: A role for CFTR

Many medications induce diarrhea as a side effect, which can be a major obstacle to therapeutic efficacy and also a life-threatening condition. Secretory diarrhea can be caused by excessive fluid secretion in the intestine under pathological conditions. The cAMP/cGMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR) is the primary chloride channel at the apical membrane of intestinal epithelial cells and plays a major role in intestinal fluid secretion and homeostasis. CFTR forms macromolecular complexes at discreet microdomains at the plasma membrane, and its chloride channel function is regulated spatiotemporally through protein-protein interactions and cAMP/cGMP-mediated signaling. Drugs that perturb CFTR-containing macromolecular complexes in the intestinal epithelium and upregulate intracellular cAMP and/or cGMP levels can hyperactivate the CFTR channel, causing excessive fluid secretion and secretory diarrhea. Inhibition of CFTR chloride-channel activity may represent a novel approach to the management of drug-induced secretory diarrhea.

Capturing the Direct Binding of CFTR Correctors to CFTR by Using Click Chemistry.

Cystic fibrosis (CF) is a lethal genetic disease caused by the loss or dysfunction of the CF transmembrane conductance regulator (CFTR) channel. F508del is the most prevalent mutation of the CFTR gene and encodes a protein defective in folding and processing. VX‐809 has been reported to facilitate the folding and trafficking of F508del‐CFTR and augment its channel function. The mechanism of action of VX‐809 has been poorly understood. In this study, we sought to answer a fundamental question underlying the mechanism of VX‐809: does it bind CFTR directly in order to exert its action? We synthesized two VX‐809 derivatives, ALK‐809 and SUL‐809, that possess an alkyne group and retain the rescue capacity of VX‐809. By using CuI‐catalyzed click chemistry, we provide evidence that the VX‐809 derivatives bind CFTR directly in vitro and in cells. Our findings will contribute to the elucidation of the mechanism of action of CFTR correctors and the design of more potent therapeutics to combat CF.

MAST205 Competes with Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-associated Ligand for Binding to CFTR to Regulate CFTR-mediated Fluid Transport

Background: CFTR is an important cAMP-regulated chloride channel. Results: MAST205 and CAL compete for binding to CFTR to regulate the expression level and function of CFTR. Conclusion: MAST205 is a regulator for CFTR. Significance: Targeting the MAST205-CFTR complex has potential clinical implications for treating CFTR-related diseases such as cystic fibrosis and secretory diarrheas. The PDZ (postsynaptic density-95/discs large/zona occludens-1) domain-based interactions play important roles in regulating the expression and function of the cystic fibrosis transmembrane conductance regulator (CFTR). Several PDZ domain-containing proteins (PDZ proteins for short) have been identified as directly or indirectly interacting with the C terminus of CFTR. To better understand the regulation of CFTR processing, we conducted a genetic screen and identified MAST205 (a microtubule-associated serine/threonine kinase with a molecular mass of 205 kDa) as a new CFTR regulator. We found that overexpression of MAST205 increased the expression of CFTR and augmented CFTR-mediated fluid transport in a dose-dependent manner. Conversely, knockdown of MAST205 inhibited CFTR function. The PDZ motif of CFTR is required for the regulatory role of MAST205 in CFTR expression and function. We further demonstrated that MAST205 and the CFTR-associated ligand competed for binding to CFTR, which facilitated the processing of CFTR and consequently up-regulated the expression and function of CFTR at the plasma membrane. More importantly, we found that MAST205 could facilitate the processing of F508del-CFTR mutant and augment its quantity and channel function at the plasma membrane. Taken together, our data suggest that MAST205 plays an important role in regulating CFTR expression and function. Our findings have important clinical implications for treating CFTR-associated diseases such as cystic fibrosis and secretory diarrheas.

Guanylate cyclase 2C agonism corrects CFTR mutants

Cystic fibrosis (CF) is a genetic disorder in which epithelium-generated fluid flow from the lung, intestine, and pancreas is impaired due to mutations disrupting CF transmembrane conductance regulator (CFTR) channel function. CF manifestations of the pancreas and lung are present in the vast majority of CF patients, and 15% of CF infants are born with obstructed gut or meconium ileus. However, constipation is a significantly underreported outcome of CF disease, affecting 47% of the CF patients, and management becomes critical in the wake of increasing life span of CF patients. In this study, we unraveled a potentially novel molecular role of a membrane-bound cyclic guanosine monophosphate-synthesizing (cGMP-synthesizing) intestinal enzyme, guanylate cyclase 2C (GCC) that could be targeted to ameliorate CF-associated intestinal fluid deficit. We demonstrated that GCC agonism results in functional rescue of murine F508del/F508del and R117H/R117H Cftr and CFTR mutants in CF patient-derived intestinal spheres. GCC coexpression and activation facilitated processing and ER exit of F508del CFTR and presented a potentially novel rescue modality in the intestine, similar to the CF corrector VX-809. Our findings identify GCC as a biological CFTR corrector and potentiator in the intestine.

Förster resonance energy transfer - an approach to visualize the spatiotemporal regulation of macromolecular complex formation and compartmentalized cell signaling.

BACKGROUND Signaling messengers and effector proteins provide an orchestrated molecular machinery to relay extracellular signals to the inside of cells and thereby facilitate distinct cellular behaviors. Formations of intracellular macromolecular complexes and segregation of signaling cascades dynamically regulate the flow of a biological process. SCOPE OF REVIEW In this review, we provide an overview of the development and application of FRET technology in monitoring cyclic nucleotide-dependent signalings and protein complexes associated with these signalings in real time and space with brief mention of other important signaling messengers and effector proteins involved in compartmentalized signaling. MAJOR CONCLUSIONS The preciseness, rapidity and specificity of cellular responses indicate restricted alterations of signaling messengers, particularly in subcellular compartments rather than globally. Not only the physical confinement and selective depletion, but also the intra- and inter-molecular interactions of signaling effectors modulate the direction of signal transduction in a compartmentalized fashion. To understand the finer details of various intracellular signaling cascades and crosstalk between proteins and other effectors, it is important to visualize these processes in live cells. Förster Resonance Energy Transfer (FRET) has been established as a useful tool to do this, even with its inherent limitations. GENERAL SIGNIFICANCE FRET technology remains as an effective tool for unraveling the complex organization and distribution of various endogenous signaling proteins, as well as the spatiotemporal dynamics of second messengers inside a single cell to distinguish the heterogeneity of cell signaling under normal physiological conditions and during pathological events.

Asymmetrical Macromolecular Complex Formation of Lysophosphatidic Acid Receptor 2 (LPA2) Mediates Gradient Sensing in Fibroblasts

Background: Chemotaxis is a fundamental process in many physiological and pathological events. Results: An LPA gradient induces a spatiotemporally restricted decrease in the mobility of LPA2 indicative of its cytoplasmic anchorage to NHERF2, the cytoskeleton and PLCβ, which causes a gradient of localized Ca2+ puffs. Conclusion: Asymmetrical macromolecular complex formation by LPA2 mediates gradient sensing. Significance: Our finding provides a new mechanistic basis to help understand chemotactic gradient sensing. Chemotactic migration of fibroblasts toward growth factors relies on their capacity to sense minute extracellular gradients and respond to spatially confined receptor-mediated signals. Currently, mechanisms underlying the gradient sensing of fibroblasts remain poorly understood. Using single-particle tracking methodology, we determined that a lysophosphatidic acid (LPA) gradient induces a spatiotemporally restricted decrease in the mobility of LPA receptor 2 (LPA2) on chemotactic fibroblasts. The onset of decreased LPA2 mobility correlates to the spatial recruitment and coupling to LPA2-interacting proteins that anchor the complex to the cytoskeleton. These localized PDZ motif-mediated macromolecular complexes of LPA2 trigger a Ca2+ puff gradient that governs gradient sensing and directional migration in response to LPA. Disruption of the PDZ motif-mediated assembly of the macromolecular complex of LPA2 disorganizes the gradient of Ca2+ puffs, disrupts gradient sensing, and reduces the directional migration of fibroblasts toward LPA. Our findings illustrate that the asymmetric macromolecular complex formation of chemoattractant receptors mediates gradient sensing and provides a new mechanistic basis for models to describe gradient sensing of fibroblasts.