scholarly journals Trout red blood cell arrestin (TRCarr), a novel member of the arrestin family: cloning, immunoprecipitation and expression of recombinant TRCarr

1996 ◽  
Vol 316 (2) ◽  
pp. 497-506 ◽  
Author(s):  
Roland JAHNS ◽  
Franck BORGESE ◽  
Sabine LINDENTHAL ◽  
Annette STRAUB ◽  
René MOTAIS ◽  
...  
Keyword(s):  
Blood Cell ◽  
Red Blood Cell ◽  
Coli Strain ◽  
G Protein Coupled Receptors ◽  
Adrenergic Stimulation ◽  
C Terminus ◽  
Protease Deficient ◽  
G Protein Coupled ◽  
Adrenergic Activation ◽  
Trout Erythrocyte

Arrestins are cytosolic proteins involved in the desensitization of G-protein-coupled receptors. We report the cloning of trout red blood cell arrestin which shows 76, 82 and 52% identity with bovine β-arrestin1, β-arrestin2 and retinal arrestin respectively. Antibodies were generated against the C-terminus of trout red blood cell arrestin. These antibodies detected arrestin in erythrocyte cytosol and were able to precipitate the native protein. The Na+/H+ antiporter of trout red blood cell is activated by β-adrenergic stimulation and is then desensitized whereas the transmembrane signalling pathway is not. To investigate the subcellular distribution of arrestin on β-adrenergic activation and desensitization of the antiporter, precipitation experiments were carried out on trout erythrocytes. A desensitization-dependent shift in cytosolic arrestin to the membranes could not be detected using the immunoprecipitation technique but we cannot exclude the possibility that a small number of cytosolic arrestins might be involved in the regulation of membrane proteins in trout erythrocyte. Recombinant trout arrestin was produced in a protease-deficient Escherichia coli strain and its functionality was tested in a reconstituted rhodopsin assay. The recombinant protein provides a suitable tool for investigating the target for arrestin in trout red blood cell, which still remains to be identified.

scholarly journals Desensitization of Adrenaline-Induced Red Blood Cell H+ Extrusion in Vitro After Chronic Exposure of Rainbow Trout to Moderate Environmental Hypoxia

1991 ◽  
Vol 156 (1) ◽  
pp. 233-248 ◽  
Author(s):  
S. THOMAS ◽  
R. KINKEAD ◽  
P. J. WALSH ◽  
C. M. WOOD ◽  
S. F. PERRY
Keyword(s):  
Rainbow Trout ◽  
Blood Cell ◽  
Red Blood Cell ◽  
Whole Blood ◽  
Inverse Correlation ◽  
Adrenergic Stimulation ◽  
Plasma Adrenaline ◽  
Arterial Blood Gas ◽  
Arterial Blood

The sensitivity of red blood cell Na+/H+ exchange to exogenous adrenaline was assessed in vitro using blood withdrawn from catheterized rainbow trout (Oncorhynchus mykiss) maintained under normoxic conditions [water PO2, (PwO2)=20.66 kPa] or after exposure to moderate hypoxia (PwO2=6.67-9.33 kPa) for 48 h, which chronically elevated plasma adrenaline, but not noradrenaline, levels. Peak changes in whole-blood extracellular pH over a 30 min period after adding 50–1000 nmoll−1 adrenaline were employed as an index of sensitivity; the blood was pre-equilibrated to simulate arterial blood gas tensions in severely hypoxic fish (PaO2=2.0 kPa, PaCO2=0.31 kPa). Blood pooled from normoxic fish displayed a dose-dependent reduction in whole-blood pH after addition of adrenaline. Blood pooled from three separate groups of hypoxic fish, however, displayed diminished sensitivity to adrenaline, ranging from complete desensitization to a 60%reduction of the response. Subsequent experiments performed on blood from individual (i.e. not pooled) normoxic or hypoxic fish demonstrated an inverse correlation between the intensity of H+ extrusion (induced by exogenous adrenaline addition) and endogenous plasma adrenaline levels at the time of blood withdrawal. However, acute increases in plasma adrenaline levels in vitro did not affect the responsiveness of the red blood cell to subsequent adrenergic stimulation. The intensity of H+ extrusion was inversely related to the PaO2in vivo between 2.67 and 10.66 kPa, and directly related to the logarithm of the endogenous plasma adrenaline level. The results suggest that desensitization of Na+/H+ exchange in chronically hypoxic fish is related to persistent elevation of levels of this catecholamine. This desensitization can be reversed in vitro as a function of time, but only when blood is maintained under sufficiently aerobic conditions.

scholarly journals Yeast-Based Directed-Evolution For High-Throughput Structural Stabilization of G Protein-Coupled Receptors (GPCRs)

2021 ◽  
Author(s):  
May Meltzer ◽  
Zvagelsky Tatiana ◽  
Niv Papo ◽  
Stanislav Engel
Keyword(s):  
G Protein ◽  
Resistant Cell ◽  
Single Step ◽  
G Protein Coupled Receptors ◽  
Structural Basis ◽  
Diffusion Method ◽  
Structural Stabilization ◽  
C Terminus ◽  
Screening Platform ◽  
G Protein Coupled

Abstract The immense potential of G protein-coupled receptors (GPCRs) as targets for drug discovery is not fully realized due to the enormous difficulties associated with structure elucidation of these profoundly unstable membrane proteins. The existing methods of GPCR stability-engineering are cumbersome and low-throughput; in addition, the scope of GPCRs that could benefit from these techniques is limited. Here, we presented a yeast-based screening platform for a single-step isolation of GRCR variants stable in the presence of short-chain detergents, a feature essential for their successful crystallization using vapor diffusion method. The detergent-resistant cell wall of yeast provides a unique compartmentalization opportunity to physically link the receptor phenotype to its encoding DNA, and thus enable discovery of stable GPCR variants with unprecedent efficiency. The scope of mutations identified by the method offers important insights into the structural basis of GPCR stability, questioning the inherent instability of the GPCR scaffold, and revealing the potential role of the C-terminus in receptor stabilization.

scholarly journals The C Terminus of the Ca Channel α1BSubunit Mediates Selective Inhibition by G-Protein-Coupled Receptors

2001 ◽  
Vol 21 (19) ◽  
pp. 7587-7597 ◽  
Author(s):  
Arthur A. Simen ◽  
Chong C. Lee ◽  
Birgitte B. Simen ◽  
Vytautas P. Bindokas ◽  
Richard J. Miller
Keyword(s):  
G Protein ◽  
Selective Inhibition ◽  
G Protein Coupled Receptors ◽  
Ca Channel ◽  
C Terminus ◽  
G Protein Coupled

scholarly journals Time course of red blood cell intracellular pH recovery following short-circuiting in relation to venous transit times in rainbow trout, Oncorhynchus mykiss

2018 ◽  
Vol 315 (2) ◽  
pp. R397-R407 ◽  
Author(s):  
Till S. Harter ◽  
Alexandra G. May ◽  
William J. Federspiel ◽  
Claudiu T. Supuran ◽  
Colin J. Brauner
Keyword(s):  
Rainbow Trout ◽  
Blood Cell ◽  
Intracellular Ph ◽  
Red Blood Cell ◽  
Time Course ◽  
Ph Sensitive ◽  
Adrenergic Stimulation ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Athletic Ability

Accumulating evidence is highlighting the importance of a system of enhanced hemoglobin-oxygen (Hb-O2) unloading for cardiovascular O2 transport in teleosts. Adrenergically stimulated sodium-proton exchangers (β-NHE) create H+ gradients across the red blood cell (RBC) membrane that are short-circuited in the presence of plasma-accessible carbonic anhydrase (paCA) at the tissues; the result is a large arterial-venous pH shift that greatly enhances O2 unloading from pH-sensitive Hb. However, RBC intracellular pH (pHi) must recover during venous transit (31–90 s) to enable O2 loading at the gills. The halftimes ( t1/2) and magnitudes of RBC β-adrenergic stimulation, short-circuiting with paCA and recovery of RBC pHi, were assessed in vitro, on rainbow trout whole blood, and using changes in closed-system partial pressure of O2 as a sensitive indicator for changes in RBC pHi. In addition, the recovery rate of RBC pHi was assessed in a continuous-flow apparatus that more closely mimics RBC transit through the circulation. Results indicate that: 1) the t1/2 of β-NHE short-circuiting is likely within the residence time of blood in the capillaries, 2) the t1/2 of RBC pHi recovery is 17 s and within the time of RBC venous transit, and 3) after short-circuiting, RBCs reestablish the initial H+ gradient across the membrane and can potentially undergo repeated cycles of short-circuiting and recovery. Thus, teleosts have evolved a system that greatly enhances O2 unloading from pH-sensitive Hb at the tissues, while protecting O2 loading at the gills; the resulting increase in O2 transport per unit of blood flow may enable the tremendous athletic ability of salmonids.

scholarly journals The Serotonin 5-HT2Aand 5-HT2CReceptors Interact with Specific Sets of PDZ Proteins

2004 ◽  
Vol 279 (19) ◽  
pp. 20257-20266 ◽  
Author(s):  
Carine Bécamel ◽  
Sophie Gavarini ◽  
Benjamin Chanrion ◽  
Gérard Alonso ◽  
Nathalie Galéotti ◽  
...  
Keyword(s):  
Protein Complexes ◽  
G Protein Coupled Receptors ◽  
Pdz Proteins ◽  
Canonical Class ◽  
Proteomic Approach ◽  
C Terminus ◽  
Intracellular Compartments ◽  
Pdz Protein ◽  
G Protein Coupled

The 5-hydroxytryptamine type 2A (5-HT2A) receptor and the 5-HT2Creceptor are closely related members of the G-protein-coupled receptors activated by serotonin that share very similar pharmacological profiles and cellular signaling pathways. These receptors express a canonical class I PDZ ligand (SXV) at their C-terminal extremity. Here, we have identified proteins that interact with the PDZ ligand of the 5-HT2Aand 5-HT2Creceptors by a proteomic approach associating affinity chromatography using immobilized synthetic peptides encompassing the PDZ ligand and mass spectrometry. We report that both receptor C termini interact with specific sets of PDZ proteinsin vitro. The 5-HT2Creceptor but not the 5-HT2Areceptor binds to the Veli-3·CASK·Mint1 ternary complex and to SAP102. In addition, the 5-HT2Creceptor binds more strongly to PSD-95 and MPP-3 than the 5-HT2Areceptor. In contrast, a robust interaction between the 5-HT2Areceptor and the channel-interacting PDZ protein CIPP was found, whereas CIPP did not significantly associate with the 5-HT2Creceptor. We also show that residues located at the -1 position and upstream the PDZ ligand in the C terminus of the 5-HT2Aand 5-HT2Creceptors are major determinants in their interaction with specific PDZ proteins. Immunofluorescence and electron microscopy studies strongly suggested that these specific interactions also take place in living cells and that the 5-HT2receptor-PDZ protein complexes occur in intracellular compartments. The interaction of the 5-HT2Aand the 5-HT2Creceptor with specific sets of PDZ proteins may contribute to their different signal transduction properties.

scholarly journals ASIC2b-dependent Regulation of ASIC3, an Essential Acid-sensing Ion Channel Subunit in Sensory Neurons via the Partner Protein PICK-1

2004 ◽  
Vol 279 (19) ◽  
pp. 19531-19539 ◽  
Author(s):  
Emmanuel Deval ◽  
Miguel Salinas ◽  
Anne Baron ◽  
Eric Lingueglia ◽  
Michel Lazdunski
Keyword(s):  
Ion Channel ◽  
Sensory Neurons ◽  
Ph Dependence ◽  
Pdz Domain ◽  
G Protein Coupled Receptors ◽  
Drg Neurons ◽  
Kinase C ◽  
C Terminus ◽  
First Time ◽  
G Protein Coupled

ASIC3, an acid-sensing ion channel subunit expressed essentially in sensory neurons, has been proposed to be involved in pain. We show here for the first time that native ASIC3-like currents were increased in cultured dorsal root ganglion (DRG) neurons following protein kinase C (PKC) stimulation. This increase was induced by the phorbol ester PDBu and by pain mediators, such as serotonin, which are known to activate the PKC pathway through their binding to G protein-coupled receptors. We demonstrate that this regulation involves the silent ASIC2b subunit, an ASIC subunit also expressed in sensory neurons. Indeed, heteromultimeric ASIC3/ASIC2b channels, but not homomeric ASIC3 channels, are positively regulated by PKC. The increase of ASIC3/ASIC2b current is accompanied by a shift in its pH dependence toward more physiological pH values and may lead to an increase of sensory neuron excitability. This regulation by PKC requires PICK-1 (protein interacting with C kinase), a PDZ domain-containing protein, which interacts with the ASIC2b C terminus.

scholarly journals Interactions of GIPC with Dopamine D2, D3 but not D4 Receptors Define a Novel Mode of Regulation of G Protein-coupled Receptors

2004 ◽  
Vol 15 (2) ◽  
pp. 696-705 ◽  
Author(s):  
Freddy Jeanneteau ◽  
Jorge Diaz ◽  
Pierre Sokoloff ◽  
Nathalie Griffon
Keyword(s):  
Rat Brain ◽  
G Protein ◽  
Plasma Membranes ◽  
Pdz Domain ◽  
G Protein Coupled Receptors ◽  
Interacting Protein ◽  
R And D ◽  
C Terminus ◽  
Endogenous Proteins ◽  
G Protein Coupled

The C-terminus domain of G protein-coupled receptors confers a functional cytoplasmic interface involved in protein association. By screening a rat brain cDNA library using the yeast two-hybrid system with the C-terminus domain of the dopamine D3 receptor (D3R) as bait, we characterized a new interaction with the PDZ domain-containing protein, GIPC (GAIP interacting protein, C terminus). This interaction was specific for the dopamine D2 receptor (D2R) and D3R, but not for the dopamine D4 receptor (D4R) subtype. Pull-down and affinity chromatography assays confirmed this interaction with recombinant and endogenous proteins. Both GIPC mRNA and protein are widely expressed in rat brain and together with the D3R in neurons of the islands of Calleja at plasma membranes and in vesicles. GIPC reduced D3R signaling, cointernalized with D2R and D3R, and sequestered receptors in sorting vesicles to prevent their lysosomal degradation. Through its dimerization, GIPC acts as a selective scaffold protein to assist receptor functions. Our results suggest a novel function for GIPC in the maintenance, trafficking, and signaling of GPCRs.

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