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Methods in Cell Biology

Arun K Shukla
No abstract text is available yet for this article.
2019: Methods in Cell Biology
Megan Gragg, Paul S-H Park
Rhodopsin is the light receptor in rod photoreceptor cells of the retina that plays a central role in phototransduction and rod photoreceptor cell health. Rhodopsin mutations are the leading known cause of autosomal dominant retinitis pigmentosa, a retinal degenerative disease. A majority of rhodopsin mutations cause misfolding and aggregation of the apoprotein opsin. The nature of aggregates formed by misfolded rhodopsin mutants and the associated cell toxicity is poorly understood. Misfolding rhodopsin mutants have been characterized biochemically, and categorized as either partial or complete misfolding mutants...
2019: Methods in Cell Biology
Renan Paulo Martin, Rafael Filippelli-Silva
G protein-coupled receptors (GPCRs) are the largest family of membrane protein playing an important role in cellular signal transduction. GPCRs interact with different molecules acting as ligands capable to trigger responses on signaling pathway. Those molecules present specific binding profiles in which, usually, are determined by methods based on radioactive labeled ligands. Here we present an alternative method based on time-resolved fluorescent labeled ligand, specific customized for angiotensin II receptors (AGTR1 and AGTR2) and kinin receptors (BDKRB1 and BDKRB2) wherein, their natural ligands were labeled with the lanthanide europium to generate the Eu3+ -N1-DTT-ligands (AngII, BK, and DBK)...
2019: Methods in Cell Biology
Holly V Shaw, Alexey Koval, Vladimir L Katanaev
Frizzleds (FZDs) are a family of GPCRs controlling key events in all branches of the developmental Wnt signaling pathway. In this capacity these receptors are mostly active prenatally and have only a limited set of functions in the human adult. Numerous cancer types and subtypes were shown to be dependent on aberrant Wnt signaling and FZDs in particular. Taken together with their GPCR properties, this makes them an attractive drug target for the development of highly specific and efficient targeted therapies against cancer...
2019: Methods in Cell Biology
Cosmo A Saunders, Ritankar Majumdar, Yaniris Molina, Bhagawat C Subramanian, Carole A Parent
Neutrophils are the most common leukocyte in human blood and are the first cells to respond to injury and infection. Improper neutrophil chemotaxis can have deleterious effects on human health, including autoimmune diseases, poor innate immune response, and cancer. Therefore, gaining a better understanding of the signaling pathways governing chemotactic responses in these cells is important. One of the main challenges of working with primary human neutrophils is their short lifespan (about 1 day), making genetic manipulations not feasible...
2019: Methods in Cell Biology
Michael J Wedemeyer, Benjamin K Mueller, Brian J Bender, Jens Meiler, Brian F Volkman
Chemokines are soluble, secreted proteins that induce chemotaxis of leukocytes and other cells. Migratory cells can sense the chemokine concentration gradient following chemokine binding and activation of chemokine receptors, a subset of the G protein-coupled receptor (GPCR) superfamily. Chemokine receptor signaling plays a central role in cell migration during inflammatory responses as well as in cancer and other diseases. Given their important role in mediating essential pathologic and physiologic processes, chemokines and their receptors are attractive targets for therapeutic development...
2019: Methods in Cell Biology
Andrew B Kleist, Francis Peterson, Robert C Tyler, Martin Gustavsson, Tracy M Handel, Brian F Volkman
The past decade has witnessed remarkable progress in the determination of G protein-coupled receptor (GPCR) structures, profoundly expanding our understanding of how GPCRs recognize ligands, become activated, and interact with intracellular signaling components. In recent years, numerous studies have used solution nuclear magnetic resonance (NMR) spectroscopy to investigate GPCRs, providing fundamental insights into GPCR conformational changes, allostery, dynamics, and other facets of GPCR function are challenging to study using other structural techniques...
2019: Methods in Cell Biology
Paula A Dos Santos Claro, Carolina Inda, Natalia G Armando, Verónica G Piazza, Alejandra Attorresi, Susana Silberstein
The development of live-cell sensors for real-time measurement of signaling responses, with improved spatial and temporal resolution with respect to classical biochemical methods, has changed our understanding of cellular signaling. Examination of cAMP generation downstream activated GPCRs has shown that signaling responses can be short-lived (generated from the cell surface) or prolonged after receptor internalization. Class B secretin-like Corticotropin-releasing hormone receptor 1 (CRHR1) is a key player in stress pathophysiology...
2019: Methods in Cell Biology
Zaira Palomino Jara, Khuraijam Dhanachandra Singh, Hamiyet Unal, Russell Desnoyer, Rodrigo Yokota, Jorge Luis Pesquero, Dulce Elena Casarini, Sadashiva S Karnik
Maintenance of normal blood pressure under conditions of drug treatment is a measure of system-wide neuro-hormonal controls and electrolyte/fluid volume homeostasis in the body. With increased interest in designing and evaluating novel drugs that may functionally select or allosterically modulate specific GPCR signaling pathways, techniques that allow us to measure acute and long-term effects on blood pressure are very important. Therefore, this chapter describes techniques to measure acute and long-term impact of novel GPCR ligands on blood pressure regulation...
2019: Methods in Cell Biology
Shelby L Wertz, Victoria L Desimine, Jennifer Maning, Katie A McCrink, Anastasios Lymperopoulos
βarrestin1 and -2 (also known as arrestin2 and -3, respectively) are G protein-coupled receptor (GPCR) adapter proteins, performing three major functions in the cell: functional desensitization, i.e., G protein uncoupling from the receptor, GPCR internalization via clathrin-coated pits, and formation of signalosomes. The βarrestins elicit a large part of the G protein-independent signaling emanating from GPCRs. Several methodologies have been developed over the past 15 years or so to quantify the GPCR-arrestin interaction/binding, especially since the latter's roles in signal transduction were discovered...
2019: Methods in Cell Biology
Gabriel Carmona-Rosas, Rocío Alcántara-Hernández, David Alejandro Hernández-Espinosa
G protein-coupled receptors (GPCRs) are transmembrane proteins that have an important impact in a myriad of cellular functions. Posttranslational modifications on GPCRs are a key processes that allow these proteins to recruit other intracellular molecules. Among these modifications, phosphorylation is the most important way of desensitization of these receptors. Several research groups have described two different desensitization mechanisms: heterologous and homologous desensitization. The first one involves the phosphorylation of the receptors by protein kinases, such as PKC, following the desensitization and internalization of the receptor, while the second one involves the phosphorylation of the receptors by GRKs, allowing for the receptor to recruit β-arrestins to be desensitized and internalized...
2019: Methods in Cell Biology
Hamiyet Unal
G protein-coupled receptors (GPCRs) play an active role in numerous cellular processes, from cell proliferation to differentiation, by modulating gene transcription through various signal transduction pathways. Transcriptional regulation coupled to reporter gene expression may be used to study both G protein-dependent and G protein-independent responses activated by GPCR ligands. Reporter genes are typically used to monitor changes in receptor-mediated cellular responses at the transcription/translation level...
2019: Methods in Cell Biology
Maarten L J Doornbos, Laura H Heitman
The superfamily of G protein-coupled receptors (GPCRs) represents the largest group of cell surface receptors in the human body. It is estimated that around 40% of the drugs currently on the market target GPCRs. As only a very small number of GPCRs is targeted by these marketed drugs, the potential of GPCRs as novel drug targets remains enormous. As opposed to conventional in vitro assays, label-free cellular assays using a biosensor provide new opportunities for studying GPCRs. Integrated receptor-mediated responses are measured in real-time rather than a single downstream signaling pathway, without the need for the use of any label (e...
2019: Methods in Cell Biology
Agata Faron-Górecka, Marta Szlachta, Magdalena Kolasa, Joanna Solich, Andrzej Górecki, Maciej Kuśmider, Dariusz Żurawek, Marta Dziedzicka-Wasylewska
Initially G protein-coupled receptors, GPCRs, were thought to act as monomers, but recently strong evidence has been gathered indicating that they are capable of forming homo- and heterodimers or higher order oligomeric complexes, and that the dimerization phenomenon can modulate the pharmacological response and function of these receptors. In this chapter we point to the great potential of alternative therapeutic approach targeted at GPCR dimers, which is especially important in the field of neuropsychopharmacology...
2019: Methods in Cell Biology
Punita Kumari, Hemlata Dwivedi, Mithu Baidya, Arun K Shukla
Agonist stimulation of G-protein-coupled receptors (GPCRs) typically results in phosphorylation and activation of ERK (Extracellular-signal Regulated Kinase) which is a member of MAP kinase (Mitogen-Activated Protein kinase) family. Detection of phosphorylated ERK1/2 MAP kinase has been widely used as readout of GPCR signaling in heterologous cells, primary cells, tissues and even in animal studies. ERK1/2 phosphorylation downstream of GPCRs is now well established to arise from the activation of both, the heterotrimeric G-proteins and β-arrestins (βarrs) with distinct spatio-temporal components...
2019: Methods in Cell Biology
Shubhi Pandey, Debarati Roy, Arun K Shukla
G protein-coupled receptors (GPCRs) constitute a large class of cell surface receptors that recognize a wide array of ligands and mediate a diverse spectrum of signaling pathways. Measuring their surface expression in cellular context is a critical aspect of studying their signaling pathways and cellular outcomes. Upon addition of agonist, GPCRs typically undergo internalization and traffic from the plasma membrane to endosomal compartments. Although radioligand binding has been the primary assay to measure GPCR surface expression and internalization, whole-cell ELISA has now emerged as a powerful alternative approach...
2019: Methods in Cell Biology
Rohan Sharma, Ravinder Gulia, Samarjit Bhattacharyya
Group I metabotropic glutamate receptors (mGluRs) are G-protein coupled receptors (GPCRs). They have been implicated in multiple forms of synaptic plasticity, as well as in various neuropsychiatric disorders. The signaling of these receptors is governed by the mechanisms of desensitization, internalization and resensitization of these receptors. Various post-translational modifications determine the signaling as well as trafficking of these receptors. Ubiquitination is a post-translational modification that has emerged as an essential regulatory process which governs group I mGluR trafficking...
2019: Methods in Cell Biology
Seung-Ryoung Jung, Bertil Hille
G protein-coupled seven-transmembrane receptors (GPCRs) mediate responses to hormones, metabolites, lipids, and neurotransmitters at the cell membrane, and so they are prominent drug targets. Although many structural, biochemical, cell biological, and biophysical studies made remarkable progress to understand mechanisms of GPCR signaling, there still are many unanswered questions about arrestin-dependent GPCR signaling. In this chapter, we focus on optical assays to see muscarinic receptor-arrestin interactions with ensemble FRET and single-molecule TIRF imaging in live cells and finally to integrate the information to simulate hypothesized steps in Virtual Cell...
2019: Methods in Cell Biology
Daniel A Fletcher, Junsang Doh, Matthieu Piel
No abstract text is available yet for this article.
2018: Methods in Cell Biology
Liang Huang, Peng Zhao, Fei Liang, Wenhui Wang
Single-cell rotation is a fundamental manipulation used in a wide range of biotechnological applications such as cell injection and enucleation. However, there are currently few methods for the 3D rotation of single cells. Here, this chapter presents different biochip platforms based on a dielectrophoresis technique to achieve 3D rotation. In-plane (yaw) and out-of-plane rotation (pitch) can be achieved by applying different AC signal configurations, respectively. This use of 3D rotation facilitates several applications...
2018: Methods in Cell Biology
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