Analysis of ciliary status via G-protein-coupled receptors localized on primary cilia

Microscopy ◽  
2020 ◽  
Vol 69 (5) ◽  
pp. 277-285
Author(s):  
Yuki Kobayashi ◽  
Akie Hamamoto ◽  
Yumiko Saito
Keyword(s):  
G Protein ◽  
Primary Cilium ◽  
Somatostatin Receptor ◽  
G Protein Coupled Receptors ◽  
The Body ◽  
Cell Surface Receptor ◽  
Specific Cell ◽  
Gpcr Signaling ◽  
Sequence Of Events ◽  
G Protein Coupled

Abstract G-protein-coupled receptors (GPCRs) comprise the largest and most diverse cell surface receptor family, with more than 800 known GPCRs identified in the human genome. Binding of an extracellular cue to a GPCR results in intracellular G protein activation, after which a sequence of events, can be amplified and optimized by selective binding partners and downstream effectors in spatially discrete cellular environments. Because GPCRs are widely expressed in the body, they help to regulate an incredible range of physiological processes from sensation to growth to hormone responses. Indeed, it is estimated that ∼ 30% of all clinically approved drugs act by binding to GPCRs. The primary cilium is a sensory organelle composed of a microtubule axoneme that extends from the basal body. The ciliary membrane is highly enriched in specific signaling components, allowing the primary cilium to efficiently convey signaling cascades in a highly ordered microenvironment. Recent data demonstrated that a limited number of non-olfactory GPCRs, including somatostatin receptor 3 and melanin-concentrating hormone receptor 1 (MCHR1), are selectively localized to cilia on several mammalian cell types including neuronal cells. Utilizing cilia-specific cell biological and molecular biological approaches, evidence has accumulated to support the biological importance of ciliary GPCR signaling followed by cilia structural changes. Thus, cilia are now considered a unique sensory platform for integration of GPCR signaling toward juxtaposed cytoplasmic structures. Herein, we review ciliary GPCRs and focus on a novel role of MCHR1 in ciliary length control that will impact ciliary signaling capacity and neuronal function.

scholarly journals Imaging of persistent cAMP signaling by internalized G protein-coupled receptors

2010 ◽  
Vol 45 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Davide Calebiro ◽  
Viacheslav O Nikolaev ◽  
Martin J Lohse
Keyword(s):  
G Protein ◽  
Intracellular Signaling ◽  
Current Model ◽  
Living Cells ◽  
G Protein Coupled Receptors ◽  
Membrane Receptors ◽  
Optical Methods ◽  
Camp Signaling ◽  
Gpcr Signaling ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) are the largest family of plasma membrane receptors. They mediate the effects of several endogenous cues and serve as important pharmacological targets. Although many biochemical events involved in GPCR signaling have been characterized in great detail, little is known about their spatiotemporal dynamics in living cells. The recent advent of optical methods based on fluorescent resonance energy transfer allows, for the first time, to directly monitor GPCR signaling in living cells. Utilizing these methods, it has been recently possible to show that the receptors for two protein/peptide hormones, the TSH and the parathyroid hormone, continue signaling to cAMP after their internalization into endosomes. This type of intracellular signaling is persistent and apparently triggers specific cellular outcomes. Here, we review these recent data and explain the optical methods used for such studies. Based on these findings, we propose a revision of the current model of the GPCR–cAMP signaling pathway to accommodate receptor signaling at endosomes.

scholarly journals Rescue of defective G protein–coupled receptor function in vivo by intermolecular cooperation

2010 ◽  
Vol 107 (5) ◽  
pp. 2319-2324 ◽  
Author(s):  
Adolfo Rivero-Müller ◽  
Yen-Yin Chou ◽  
Inhae Ji ◽  
Svetlana Lajic ◽  
Aylin C. Hanyaloglu ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Luteinizing Hormone Receptor ◽  
Wild Type ◽  
Gpcr Signaling ◽  
Physiological Relevance ◽  
Biosynthesis Activation ◽  
Mutant Receptors ◽  
G Protein Coupled

G protein–coupled receptors (GPCRs) are ubiquitous mediators of signaling of hormones, neurotransmitters, and sensing. The old dogma is that a one ligand/one receptor complex constitutes the functional unit of GPCR signaling. However, there is mounting evidence that some GPCRs form dimers or oligomers during their biosynthesis, activation, inactivation, and/or internalization. This evidence has been obtained exclusively from cell culture experiments, and proof for the physiological significance of GPCR di/oligomerization in vivo is still missing. Using the mouse luteinizing hormone receptor (LHR) as a model GPCR, we demonstrate that transgenic mice coexpressing binding-deficient and signaling-deficient forms of LHR can reestablish normal LH actions through intermolecular functional complementation of the mutant receptors in the absence of functional wild-type receptors. These results provide compelling in vivo evidence for the physiological relevance of intermolecular cooperation in GPCR signaling.

scholarly journals Beyond the Flavour: The Potential Druggability of Chemosensory G Protein-Coupled Receptors

2019 ◽  
Vol 20 (6) ◽  
pp. 1402 ◽  
Author(s):  
Antonella Di Pizio ◽  
Maik Behrens ◽  
Dietmar Krautwurst
Keyword(s):  
G Protein ◽  
Drug Targets ◽  
Current Knowledge ◽  
Chemical Space ◽  
G Protein Coupled Receptors ◽  
The Body ◽  
Odorant Receptors ◽  
Taste Receptors ◽  
Umami Taste ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) belong to the largest class of drug targets. Approximately half of the members of the human GPCR superfamily are chemosensory receptors, including odorant receptors (ORs), trace amine-associated receptors (TAARs), bitter taste receptors (TAS2Rs), sweet and umami taste receptors (TAS1Rs). Interestingly, these chemosensory GPCRs (csGPCRs) are expressed in several tissues of the body where they are supposed to play a role in biological functions other than chemosensation. Despite their abundance and physiological/pathological relevance, the druggability of csGPCRs has been suggested but not fully characterized. Here, we aim to explore the potential of targeting csGPCRs to treat diseases by reviewing the current knowledge of csGPCRs expressed throughout the body and by analysing the chemical space and the drug-likeness of flavour molecules.

scholarly journals A Homogeneous Enzyme Fragment Complementation-Based β-Arrestin Translocation Assay for High-Throughput Screening of G-Protein-Coupled Receptors

2008 ◽  
Vol 13 (8) ◽  
pp. 737-747 ◽  
Author(s):  
Xiaoning Zhao ◽  
Adrie Jones ◽  
Keith R. Olson ◽  
Kun Peng ◽  
Tom Wehrman ◽  
...  
Keyword(s):  
G Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Somatostatin Receptor ◽  
Gene Families ◽  
G Protein Coupled Receptors ◽  
Functional Assay ◽  
Gpcr Activation ◽  
Fragment Complementation ◽  
G Protein Coupled

G-protein-coupled receptors (GPCRs) represent one of the largest gene families in the human genome and have long been regarded as valuable targets for small-molecule drugs. The authors describe a new functional assay that directly monitors GPCR activation. It is based on the interaction between β-arrestin and ligand-activated GPCRs and uses enzyme fragment complementation technology. In this format, a GPCR of interest is fused to a small (~4 kDa), optimized α fragment peptide (termed ProLink™) derived from β-galactosidase, and β-arrestin is fused to an N-terminal deletion mutant of β-galactosidase (termed the enzyme acceptor [EA]). Upon activation of the receptor, the β-arrestin-EA fusion protein binds the activated GPCR. This interaction drives enzyme fragment complementation, resulting in an active β-galactosidase enzyme, and thus GPCR activation can be determined by quantifying β-galactosidase activity. In this report, the authors demonstrate the utility of this technology to monitor GPCR activation and validate the approach using a Gαi-coupled GPCR, somatostatin receptor 2. Potential application to high-throughput screens in both agonist and antagonist screening modes is exemplified. ( Journal of Biomolecular Screening 2008:737-747)

scholarly journals An Intracellular Loop (IL2) Residue Confers Different Basal Constitutive Activities to the Human Lutropin Receptor and Human Thyrotropin Receptor through Structural Communication between IL2 and Helix 6, via Helix 3

Endocrinology ◽  
2007 ◽  
Vol 149 (4) ◽  
pp. 1705-1717 ◽  
Author(s):  
Xiuyan Feng ◽  
Thomas Müller ◽  
Dario Mizrachi ◽  
Francesca Fanelli ◽  
Deborah L. Segaloff
Keyword(s):  
G Protein ◽  
Structural Model ◽  
G Protein Coupled Receptors ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Solvent Exposure ◽  
Lutropin Receptor ◽  
Reciprocal Arrangement ◽  
G Protein Coupled ◽  
Constitutively Active

The human lutropin receptor (hLHR) and human TSH receptor (hTSHR) are G protein-coupled receptors that play key roles in reproductive and thyroid physiology, respectively. We show using a quantitative assessment of cAMP production as a function of cell surface receptor expression that the hTSHR possesses greater basal constitutive activity than the hLHR. Further studies were undertaken to test the hypothesis that different potential Gs-coupling motifs identified in IL2 of the hTSHR and hLHR contribute to their different basal constitutive activities. Although mutating the receptors to interchange their potential Gs-coupling motifs reversed their relative activities, we show this to be due to the swapping of one IL2 residue (Q476 in the hLHR; R531 in the hTSHR). Molecular dynamics simulations show that the effect of the hLHR(Q476R) mutation, switching the structural features of the hLHR toward those of the hTSHR, is greater than the switching effect of the hTSHR(R531Q) mutant toward the hLHR. The structural model of the hLHR(Q476R) mutant can be considered as a hybrid of wild-type (wt) hTSHR and constitutively active mutant hLHR forms. In this hLHR(Q476R) mutant, IL2 adopts a structure similar to IL2 of the wt hTSHR, but it shares with the hLHR constitutively active mutant the solvent exposure and the reciprocal arrangement of helices 3, 5, and 6, including the weakening of the wt native R3.50-D6.30 interaction. Our results suggest a H3-mediated structural connection between IL2 and the cytosolic extension of H6. Thus, IL2 contributes significantly to the inactive and active state ensembles of these G protein-coupled receptors.

scholarly journals Mycobacteria Manipulate G-Protein-Coupled Receptors to Increase Mucosal Rac1 Expression in the Lungs

2016 ◽  
Vol 9 (3) ◽  
pp. 318-329 ◽  
Author(s):  
Nader Alaridah ◽  
Nataliya Lutay ◽  
Erik Tenland ◽  
Anna Rönnholm ◽  
Oskar Hallgren ◽  
...  
Keyword(s):  
G Protein ◽  
Airway Epithelial Cells ◽  
G Protein Coupled Receptors ◽  
The Body ◽  
Airway Epithelial ◽  
Mucosal Vaccination ◽  
Mapk Signalling Pathway ◽  
The Impact ◽  
G Protein Coupled ◽  
Rac1 Expression

Mycobacterium bovis bacille Calmette-Guérin (BCG) is currently the only approved vaccine against tuberculosis (TB). BCG mimics M. tuberculosis (Mtb) in its persistence in the body and is used as a benchmark to compare new vaccine candidates. BCG was originally designed for mucosal vaccination, but comprehensive knowledge about its interaction with epithelium is currently lacking. We used primary airway epithelial cells (AECs) and a murine model to investigate the initial events of mucosal BCG interactions. Furthermore, we analysed the impact of the G-protein-coupled receptors (GPCRs), CXCR1 and CXCR2, in this process, as these receptors were previously shown to be important during TB infection. BCG infection of AECs induced GPCR-dependent Rac1 up-regulation, resulting in actin redistribution. The altered distribution of the actin cytoskeleton involved the MAPK signalling pathway. Blocking of the CXCR1 or CXCR2 prior to infection decreased Rac1 expression, and increased epithelial transcriptional activity and epithelial cytokine production. BCG infection did not result in epithelial cell death as measured by p53 phosphorylation and annexin. This study demonstrated that BCG infection of AECs manipulated the GPCRs to suppress epithelial signalling pathways. Future vaccine strategies could thus be improved by targeting GPCRs.

scholarly journals Structural insights into emergent signaling modes of G protein–coupled receptors

2020 ◽  
Vol 295 (33) ◽  
pp. 11626-11642 ◽  
Author(s):  
Ieva Sutkeviciute ◽  
Jean-Pierre Vilardaga
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Prescription Drugs ◽  
Helical Structure ◽  
G Protein Coupled Receptors ◽  
Gpcr Signaling ◽  
Gpcr Activation ◽  
Endosomal Membranes ◽  
G Protein Coupled ◽  
Structural Insights

G protein–coupled receptors (GPCRs) represent the largest family of cell membrane proteins, with >800 GPCRs in humans alone, and recognize highly diverse ligands, ranging from photons to large protein molecules. Very important to human medicine, GPCRs are targeted by about 35% of prescription drugs. GPCRs are characterized by a seven-transmembrane α-helical structure, transmitting extracellular signals into cells to regulate major physiological processes via heterotrimeric G proteins and β-arrestins. Initially viewed as receptors whose signaling via G proteins is delimited to the plasma membrane, it is now recognized that GPCRs signal also at various intracellular locations, and the mechanisms and (patho)physiological relevance of such signaling modes are actively investigated. The propensity of GPCRs to adopt different signaling modes is largely encoded in the structural plasticity of the receptors themselves and of their signaling complexes. Here, we review emerging modes of GPCR signaling via endosomal membranes and the physiological implications of such signaling modes. We further summarize recent structural insights into mechanisms of GPCR activation and signaling. We particularly emphasize the structural mechanisms governing the continued GPCR signaling from endosomes and the structural aspects of the GPCR resensitization mechanism and discuss the recently uncovered and important roles of lipids in these processes.

G-protein-coupled receptors, channels, and Na+–H+ exchanger in nuclear membranes of heart, hepatic, vascular endothelial, and smooth muscle cellsThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.

2006 ◽  
Vol 84 (3-4) ◽  
pp. 431-441 ◽  
Author(s):  
Ghassan Bkaily ◽  
Moni Nader ◽  
Levon Avedanian ◽  
Sana Choufani ◽  
Danielle Jacques ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
G Protein ◽  
Cell Types ◽  
G Protein Coupled Receptors ◽  
Specific Cell ◽  
Endothelin 1 ◽  
Nuclear Membranes ◽  
Ionic Transporters ◽  
A Cell ◽  
G Protein Coupled

The action of several peptides and drugs is thought to be primarily dependent on their interactions with specific cell surface G-protein-coupled receptors and ionic transporters such as channels and exchangers. Recent development of 3-D confocal microscopy allowed several laboratories, including ours, to identify and study the localization of receptors, channels, and exchangers at the transcellular level of several cell types. Using this technique, we demonstrated in the nuclei of several types of cells the presence of Ca2+ channels as well as Na+–H+ exchanger and receptors such as endothelin-1 and angiotensin II receptors. Stimulation of these nuclear membrane G-protein-coupled receptors induced an increase of nuclear Ca2+. Our results suggest that, similar to the plasma membrane, nuclear membranes possess channels, exchangers and receptors such as those for endothelin-1 and angiotensin II, and that the nucleus seems to be a cell within a cell. This article will emphasize these findings.

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