scholarly journals Enlightening G-protein-coupled receptors on the plasma membrane using super-resolution photoactivated localization microscopy

2013 ◽  
Vol 41 (1) ◽  
pp. 191-196 ◽  
Author(s):  
Marco Scarselli ◽  
Paolo Annibale ◽  
Claudio Gerace ◽  
Aleksandra Radenovic
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Super Resolution ◽  
Molecular Size ◽  
G Protein Coupled Receptors ◽  
Temporal Organization ◽  
Localization Microscopy ◽  
Photoactivated Localization Microscopy ◽  
Super Resolution Microscopy ◽  
G Protein Coupled

The possibility to visualize and image the arrangement of proteins within the cell at the molecular level has always been an attraction for scientists in biological research. In particular, for signalling molecules such as GPCRs (G-protein-coupled receptors), the existence of protein aggregates such as oligomers or clusters has been the topic of extensive debate. One of the reasons for this lively argument is that the molecular size is below the diffraction-limited resolution of the conventional microscopy, precluding the direct visualization of protein super-structures. On the other hand, new super-resolution microscopy techniques, such as the PALM (photoactivated localization microscopy), allow the limit of the resolution power of conventional optics to be broken and the localization of single molecules to be determined with a precision of 10–20 nm, close to their molecular size. The application of super-resolution microscopy to study the spatial and temporal organization of GPCRs has brought new insights into receptor arrangement on the plasma membrane. Furthermore, the use of this powerful microscopy technique as a quantitative tool opens up the possibility for investigating and quantifying the number of molecules in biological assemblies and determining the protein stoichiometry in signalling complexes.

scholarly journals Molecular Signatures of G-Protein Coupled Receptors in Pancreatic Cancer using Super-Resolution Microscopy

2015 ◽  
Vol 108 (2) ◽  
pp. 96a
Author(s):  
Raphael Jorand ◽  
Ottavia Golfetto ◽  
Sunetra Biswas ◽  
Steven J. Tobin ◽  
Huiying Zhang ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
G Protein ◽  
Super Resolution ◽  
G Protein Coupled Receptors ◽  
Molecular Signatures ◽  
Super Resolution Microscopy ◽  
G Protein Coupled

scholarly journals Misfolded G Protein-Coupled Receptors and Endocrine Disease. Molecular Mechanisms and Therapeutic Prospects

2021 ◽  
Vol 22 (22) ◽  
pp. 12329
Author(s):  
Alfredo Ulloa-Aguirre ◽  
Teresa Zariñán ◽  
Eduardo Jardón-Valadez
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Intracellular Signaling ◽  
Molecular Mechanisms ◽  
G Protein Coupled Receptors ◽  
Membrane Receptors ◽  
Therapeutic Interventions ◽  
Endocrine Function ◽  
Receptor Protein ◽  
G Protein Coupled

Misfolding of G protein-coupled receptors (GPCRs) caused by mutations frequently leads to disease due to intracellular trapping of the conformationally abnormal receptor. Several endocrine diseases due to inactivating mutations in GPCRs have been described, including X-linked nephrogenic diabetes insipidus, thyroid disorders, familial hypocalciuric hypercalcemia, obesity, familial glucocorticoid deficiency [melanocortin-2 receptor, MC2R (also known as adrenocorticotropin receptor, ACTHR), and reproductive disorders. In these mutant receptors, misfolding leads to endoplasmic reticulum retention, increased intracellular degradation, and deficient trafficking of the abnormal receptor to the cell surface plasma membrane, causing inability of the receptor to interact with agonists and trigger intracellular signaling. In this review, we discuss the mechanisms whereby mutations in GPCRs involved in endocrine function in humans lead to misfolding, decreased plasma membrane expression of the receptor protein, and loss-of-function diseases, and also describe several experimental approaches employed to rescue trafficking and function of the misfolded receptors. Special attention is given to misfolded GPCRs that regulate reproductive function, given the key role played by these particular membrane receptors in sexual development and fertility, and recent reports on promising therapeutic interventions targeting trafficking of these defective proteins to rescue completely or partially their normal function.

scholarly journals Actin filaments partition primary cilia membranes into distinct fluid corrals

2018 ◽  
Vol 217 (8) ◽  
pp. 2831-2849 ◽  
Author(s):  
Sungsu Lee ◽  
Han Yen Tan ◽  
Ivayla I. Geneva ◽  
Aleksandr Kruglov ◽  
Peter D. Calvert
Keyword(s):  
Diffusion Coefficient ◽  
G Protein ◽  
Primary Cilia ◽  
Super Resolution ◽  
G Protein Coupled Receptors ◽  
Membrane Diffusion ◽  
Filamentous Actin ◽  
Spatiotemporal Resolution ◽  
Fast Tracking ◽  
G Protein Coupled

Physical properties of primary cilia membranes in living cells were examined using two independent, high-spatiotemporal-resolution approaches: fast tracking of single quantum dot–labeled G protein–coupled receptors and a novel two-photon super-resolution fluorescence recovery after photobleaching of protein ensemble. Both approaches demonstrated the cilium membrane to be partitioned into corralled domains spanning 274 ± 20 nm, within which the receptors are transiently confined for 0.71 ± 0.09 s. The mean membrane diffusion coefficient within the corrals, Dm1 = 2.9 ± 0.41 µm2/s, showed that the ciliary membranes were among the most fluid encountered. At longer times, the apparent membrane diffusion coefficient, Dm2 = 0.23 ± 0.05 µm2/s, showed that corral boundaries impeded receptor diffusion 13-fold. Mathematical simulations predict the probability of G protein–coupled receptors crossing corral boundaries to be 1 in 472. Remarkably, latrunculin A, cytochalasin D, and jasplakinolide treatments altered the corral permeability. Ciliary membranes are thus partitioned into highly fluid membrane nanodomains that are delimited by filamentous actin.

scholarly journals Selectivity Landscape of 100 Therapeutically Relevant GPCR Profiled by an Effector Translocation-Based BRET Platform

2020 ◽  
Author(s):  
Charlotte Avet ◽  
Arturo Mancini ◽  
Billy Breton ◽  
Christian Le Gouill ◽  
Alexander S. Hauser ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Drug Discovery ◽  
Signaling Pathways ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Systems Pharmacology ◽  
New Knowledge ◽  
Translocation Assay ◽  
Multiple Signaling ◽  
G Protein Coupled

SUMMARYThe ability of individual G protein-coupled receptors (GPCR) to engage multiple signaling pathways opens opportunities for the development of better drugs. This requires new knowledge and tools to determine the G protein subtypes and βarrestins engaged by a given receptor. Here, we used a new BRET-based effector membrane translocation assay (EMTA) that monitors activation of each Gα protein through the recruitment of selective G protein effectors and βarrestins to the plasma membrane. Profiling of 100 therapeutically relevant GPCR revealed a great diversity of coupling profiles with some receptors displaying exquisite selectivity, whereas others promiscuitely engage all four G protein families. Comparison with existing datasets points to commonalities but also to critical differences between studies. Combining a biosensor subset allowed detecting activity of nearly all GPCR thus providing a new tool for safety screens and systems pharmacology. Overall, this work describes unique resources for studying GPCR function and drug discovery.

scholarly journals Organized cannabinoid receptor distribution in neurons revealed by super-resolution fluorescence imaging

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Hui Li ◽  
Jie Yang ◽  
Cuiping Tian ◽  
Min Diao ◽  
Quan Wang ◽  
...  
Keyword(s):  
G Protein ◽  
Fluorescence Imaging ◽  
Cannabinoid Receptor ◽  
Super Resolution ◽  
G Protein Coupled Receptors ◽  
Cellular Functions ◽  
Cannabinoid Receptor 1 ◽  
Intracellular Organization ◽  
Dynamic Movement ◽  
G Protein Coupled

Abstract G-protein-coupled receptors (GPCRs) play important roles in cellular functions. However, their intracellular organization is largely unknown. Through investigation of the cannabinoid receptor 1 (CB1), we discovered periodically repeating clusters of CB1 hotspots within the axons of neurons. We observed these CB1 hotspots interact with the membrane-associated periodic skeleton (MPS) forming a complex crucial in the regulation of CB1 signaling. Furthermore, we found that CB1 hotspot periodicity increased upon CB1 agonist application, and these activated CB1 displayed less dynamic movement compared to non-activated CB1. Our results suggest that CB1 forms periodic hotspots organized by the MPS as a mechanism to increase signaling efficacy upon activation.

Targeting of cytosolic phospholipase A2 to plasma membrane inhibits its activation by G-protein coupled receptors

Lipids ◽  
1999 ◽  
Vol 34 (S1) ◽  
pp. S67-S67
Author(s):  
Elsa Klapisz ◽  
Mouloud Ziari ◽  
Kamen Koumanov ◽  
Dominique Wendum ◽  
Corinne Brachet ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phospholipase A2 ◽  
G Protein ◽  
Cytosolic Phospholipase A2 ◽  
G Protein Coupled Receptors ◽  
Cytosolic Phospholipase ◽  
G Protein Coupled

scholarly journals Identification of G Protein α Subunit-Palmitoylating Enzyme

2008 ◽  
Vol 29 (2) ◽  
pp. 435-447 ◽  
Author(s):  
Ryouhei Tsutsumi ◽  
Yuko Fukata ◽  
Jun Noritake ◽  
Tsuyoshi Iwanaga ◽  
Franck Perez ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
G Protein ◽  
Adrenergic Receptor ◽  
Fluorescence Recovery After Photobleaching ◽  
G Protein Coupled Receptors ◽  
Α Subunit ◽  
Cytoplasmic Face ◽  
G Protein Α Subunit ◽  
G Protein Coupled

ABSTRACT The heterotrimeric G protein α subunit (Gα) is targeted to the cytoplasmic face of the plasma membrane through reversible lipid palmitoylation and relays signals from G-protein-coupled receptors (GPCRs) to its effectors. By screening 23 DHHC motif (Asp-His-His-Cys) palmitoyl acyl-transferases, we identified DHHC3 and DHHC7 as Gα palmitoylating enzymes. DHHC3 and DHHC7 robustly palmitoylated Gαq, Gαs, and Gαi2 in HEK293T cells. Knockdown of DHHC3 and DHHC7 decreased Gαq/11 palmitoylation and relocalized it from the plasma membrane into the cytoplasm. Photoconversion analysis revealed that Gαq rapidly shuttles between the plasma membrane and the Golgi apparatus, where DHHC3 specifically localizes. Fluorescence recovery after photobleaching studies showed that DHHC3 and DHHC7 are necessary for this continuous Gαq shuttling. Furthermore, DHHC3 and DHHC7 knockdown blocked the α1A-adrenergic receptor/Gαq/11-mediated signaling pathway. Together, our findings revealed that DHHC3 and DHHC7 regulate GPCR-mediated signal transduction by controlling Gα localization to the plasma membrane.

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