Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of ...

Cincinnati Children’s Hospital Medical Center

University of Tennessee Health Science Center

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.

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

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.

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

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 Ca(2+) 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 Ca(2+) 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.

Asymmetrical macromolecular complex formation of lysophosphatidic acid receptor 2 (LPA2) mediates gradient sensing in fibroblasts.

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.

Compartmentalized accumulation of cAMP near complexes of multidrug resistance protein 4 (MRP4) and cystic fibrosis transmembrane conductance regulator (CFTR) contributes to drug-induced diarrhea.

Multidrug resistance protein 4 (MRP4), a member of the ATP binding cassette transporter family, functions as a plasma membrane exporter of cyclic nucleotides. Recently, we demonstrated that fibroblasts lacking the Mrp4 gene migrate faster and contain higher cyclic-nucleotide levels. Here, we show that cAMP accumulation and protein kinase A (PKA) activity are higher and polarized in Mrp4(-/-) fibroblasts, versus Mrp4(+/+) cells. MRP4-containing macromolecular complexes isolated from these fibroblasts contained several proteins, including actin, which play important roles in cell migration. We found that actin interacts with MRP4, predominantly at the plasma membrane, and an intact actin cytoskeleton is required to restrict MRP4 to specific microdomains of the plasma membrane. Our data further indicated that the enhanced accumulation of cAMP in Mrp4(-/-) fibroblasts facilitates cortical actin polymerization in a PKA-dependent manner at the leading edge, which in turn increases the overall rate of cell migration to accelerate the process of wound healing. Disruption of actin polymerization or inhibition of PKA activity abolished the effect of MRP4 on cell migration. Together, our findings suggest a novel cAMP-dependent mechanism for MRP4-mediated regulation of fibroblast migration whereby PKA and actin play critical roles as downstream effectors.

PKA and actin play critical roles as downstream effectors in MRP4-mediated regulation of fibroblast migration.

Core binding factor (CBF) is a heterodimeric protein complex involved in the transcriptional regulation of normal hematopoiesis. Mutations in CBF-encoding genes result in leukemogenic proliferative advantages and impaired differentiation of the hematopoietic progenitors. CBF molecular aberrations are responsible for approximately 20% of all adult acute myeloid leukemia (AML). Although CBF-AMLs are considered to have relatively good prognosis compared to other leukemia subtypes, they are a heterogeneous group of disorders and modern therapy frequently leads to relapse and the associated morbidity and mortality. Improvements in risk stratification and development of targeted therapies are needed for better outcomes. In this review we provide a brief overview of the molecular basis, prognostic categories and the advanced treatment strategies for CBF leukemias.

Core Binding Factor Acute Myeloid Leukemia: New Prognostic Categories and Therapeutic Opportunities.

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 Cu(I) -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.

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

Ulcerative colitis (UC) belongs to inflammatory bowel disorders, a group of gastrointestinal disorders that can produce serious recurring diarrhea in affected patients. The mechanism for UC- and inflammatory bowel disorder-associated diarrhea is not well understood. The cystic fibrosis transmembrane-conductance regulator (CFTR) chloride channel plays an important role in fluid and water transport across the intestinal mucosa. CFTR channel function is regulated in a compartmentalized manner through the formation of CFTR-containing macromolecular complexes at the plasma membrane. In this study, we demonstrate the involvement of a novel macromolecular signaling pathway that causes diarrhea in UC. We found that a nitric oxide-producing enzyme, inducible nitric oxide synthase (iNOS), is overexpressed under the plasma membrane and generates compartmentalized cGMP in gut epithelia in UC. The scaffolding protein Na(+)/H(+) exchanger regulatory factor 2 (NHERF2) bridges iNOS with CFTR, forming CFTR-NHERF2-iNOS macromolecular complexes that potentiate CFTR channel function via the nitric oxide-cGMP pathway under inflammatory conditions both in vitro and in vivo. Potential disruption of these complexes in Nherf2(-/-) mice may render them more resistant to CFTR-mediated secretory diarrhea than Nherf2(+/+) mice in murine colitis models. Our study provides insight into the mechanism of pathophysiologic occurrence of diarrhea in UC and suggests that targeting CFTR and CFTR-containing macromolecular complexes will ameliorate diarrheal symptoms and improve conditions associated with inflammatory bowel disorders.

Chandrima Sinha

Division of Pulmonary Medicine,
Department of Pediatrics,
Division of Pulmonary Medicine, Department of Pediatrics

Cincinnati Children’s Hospital Medical Center

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Chandrima Sinha

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Pulmonary Medicine,Department of Pediatrics,Division of Pulmonary Medicine, Department of Pediatrics

Cincinnati Children’s Hospital Medical Center

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Division of Pulmonary Medicine,
Department of Pediatrics,
Division of Pulmonary Medicine, Department of Pediatrics