Assessing Cyclic Nucleotide Recognition in Cells: Opportunities and Pitfalls for Selective Receptor Activation

2015 ◽  
pp. 78-97
Keyword(s):  
Cyclic Nucleotide ◽  
Receptor Activation ◽  
Nucleotide Recognition ◽  
In Cells

scholarly journals G-CSF receptor activation of the Src kinase Lyn is mediated by Gab2 recruitment of the Shp2 phosphatase

Blood ◽  
2011 ◽  
Vol 118 (4) ◽  
pp. 1077-1086 ◽  
Author(s):  
Muneyoshi Futami ◽  
Quan-sheng Zhu ◽  
Zakary L. Whichard ◽  
Ling Xia ◽  
Yuehai Ke ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Src Kinase ◽  
Receptor Activation ◽  
Phosphorylation Sites ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Stimulating Factor ◽  
In Cells

Abstract Src activation involves the coordinated regulation of positive and negative tyrosine phosphorylation sites. The mechanism whereby receptor tyrosine kinases, cytokine receptors, and integrins activate Src is not known. Here, we demonstrate that granulocyte colony-stimulating factor (G-CSF) activates Lyn, the predominant Src kinase in myeloid cells, through Gab2-mediated recruitment of Shp2. After G-CSF stimulation, Lyn dynamically associates with Gab2 in a spatiotemporal manner. The dephosphorylation of phospho-Lyn Tyr507 was abrogated in Shp2-deficient cells transfected with the G-CSF receptor but intact in cells expressing phosphatase-defective Shp2. Auto-phosphorylation of Lyn Tyr396 was impaired in cells treated with Gab2 siRNA. The constitutively activated Shp2E76A directed the dephosphorylation of phospho-Lyn Tyr507 in vitro. Tyr507 did not undergo dephosphorylation in G-CSF–stimulated cells expressing a mutant Gab2 unable to bind Shp2. We propose that Gab2 forms a complex with Lyn and after G-CSF stimulation, Gab2 recruits Shp2, which dephosphorylates phospho-Lyn Tyr507, leading to Lyn activation.

The first 3′:5′-cyclic nucleotide–amino acid complex:L-His–cIMP

2012 ◽  
Vol 68 (8) ◽  
pp. o311-o316 ◽  
Author(s):  
Katarzyna Ślepokura
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Cyclic Nucleotide ◽  
Phosphate Group ◽  
Specific Amino Acid ◽  
Π Stacking ◽  
Cyclic Phosphate ◽  
Nucleotide Recognition ◽  
Group Play ◽  
Phosphate Groups

In the crystal structure of the L-His–cIMP complex,i.e.L-histidinium inosine 3′:5′-cyclic phosphate [systematic name: 5-(2-amino-2-carboxyethyl)-1H-imidazol-3-ium 7-hydroxy-2-oxo-6-(6-oxo-6,9-dihydro-1H-purin-9-yl)-4a,6,7,7a-tetrahydro-4H-1,3,5,2λ5-furo[3,2-d][1,3,2λ5]dioxaphosphinin-2-olate], C6H10N3O2+·C10H10N4O7P−, the Hoogsteen edge of the hypoxanthine (Hyp) base of cIMP and the Hyp face are engaged in specific amino acid–nucleotide (His...cIMP) recognition,i.e.by abutting edge-to-edge and by π–π stacking, respectively. The Watson–Crick edge of Hyp and the cIMP phosphate group play a role in nonspecific His...cIMP contacts. The interactions between the cIMP anions (anti/C3′–endo/trans–gauche/chair conformers) are realized mainly between riboses and phosphate groups. The results for this L-His–cIMP complex, compared with those for the previously reported solvated L-His–IMP crystal structure, indicate a different nature of amino acid–nucleotide recognition and interactions upon the 3′:5′-cyclization of the nucleotide phosphate group.

Development and validation of algorithms for measuring G-protein coupled receptor activation in cells using the LSC-based imaging cytometer platform

2005 ◽  
Vol 65A (1) ◽  
pp. 69-76 ◽  
Author(s):  
Kazuo Ozawa ◽  
Christine C. Hudson ◽  
Kirsten R. Wille ◽  
Sachiko Karaki ◽  
Robert H. Oakley
Keyword(s):  
G Protein ◽  
Receptor Activation ◽  
G Protein Coupled Receptor ◽  
Development And Validation ◽  
G Protein Coupled ◽  
In Cells

scholarly journals PDGF receptor-β modulates metanephric mesenchyme chemotaxis induced by PDGF AA

2009 ◽  
Vol 296 (2) ◽  
pp. F406-F417 ◽  
Author(s):  
Jill M. Ricono ◽  
Brent Wagner ◽  
Yves Gorin ◽  
Mazen Arar ◽  
Andrius Kazlauskas ◽  
...  
Keyword(s):  
Cell Migration ◽  
Kidney Development ◽  
Receptor Activation ◽  
Ros Generation ◽  
Pdgf Receptor ◽  
Metanephric Mesenchyme ◽  
Wild Type ◽  
Intracellular Ca ◽  
Β Receptor ◽  
In Cells

PDGF B chain or PDGF receptor (PDGFR)-β-deficient (−/−) mice lack mesangial cells. To study responses of α- and β-receptor activation to PDGF ligands, metanephric mesenchymal cells (MMCs) were established from embryonic day E11.5 wild-type (+/+) and −/− mouse embryos. PDGF BB stimulated cell migration in +/+ cells, whereas PDGF AA did not. Conversely, PDGF AA was chemotactic for −/− MMCs. The mechanism by which PDGFR-β inhibited AA-induced migration was investigated. PDGF BB, but not PDGF AA, increased intracellular Ca2+ and the production of reactive oxygen species (ROS) in +/+ cells. Transfection of −/− MMCs with the wild-type β-receptor restored cell migration and ROS generation in response to PDGF BB and inhibited AA-induced migration. Inhibition of Ca2+ signaling facilitated PDGF AA-induced chemotaxis in the wild-type cells. The antioxidant N-acetyl-l-cysteine (NAC) or the NADPH oxidase inhibitor diphenyleneiodonium (DPI) abolished the BB-induced increase in intracellular Ca2+ concentration, suggesting that ROS act as upstream mediators of Ca2+ in suppressing PDGF AA-induced migration. These data indicate that ROS and Ca2+ generated by active PDGFR-β play an essential role in suppressing PDGF AA-induced migration in +/+ MMCs. During kidney development, PDGFR β-mediated ROS generation and Ca2+ influx suppress PDGF AA-induced chemotaxis in metanephric mesenchyme.

scholarly journals ETB receptor activation leads to activation and phosphorylation of NHE3

1999 ◽  
Vol 276 (4) ◽  
pp. C938-C945 ◽  
Author(s):  
Y. Peng ◽  
O. W. Moe ◽  
T.-S. Chu ◽  
P. A. Preisig ◽  
M. Yanagisawa ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Tyrosine Phosphorylation ◽  
Time Course ◽  
Receptor Activation ◽  
Endothelin Receptors ◽  
Receptor Specificity ◽  
Low Concentrations ◽  
Sds Page ◽  
Etb Receptor ◽  
In Cells

In OKP cells expressing ETB endothelin receptors, activation of Na+/H+antiporter activity by endothelin-1 (ET-1) was resistant to low concentrations of ethylisopropyl amiloride, indicating regulation of Na+/H+exchanger isoform 3 (NHE3). ET-1 increased NHE3 phosphorylation in cells expressing ETB receptors but not in cells expressing ETAreceptors. Receptor specificity was not due to demonstrable differences in receptor-specific activation of tyrosine phosphorylation pathways or inhibition of adenylyl cyclase. Phosphorylation was associated with a decrease in mobility on SDS-PAGE, which was reversed by treating immunoprecipitated NHE3 with alkaline phosphatase. Phosphorylation was first seen at 5 min and was maximal at 15–30 min. Phosphorylation was maximal with 10−9 M ET-1. Phosphorylation occurred on threonine and serine residues at multiple sites. In summary, ET-1 induces NHE3 phosphorylation in OKP cells on multiple threonine and serine residues. ETB receptor specificity, time course, and concentration dependence are all similar between ET-1-induced increases in NHE3 activity and phosphorylation, suggesting that phosphorylation plays a key role in activation.

Cyclic Nucleotide Phosphodiesterase Activity, Expression, and Targeting in Cells of the Cardiovascular System

2003 ◽  
Vol 64 (3) ◽  
pp. 533-546 ◽  
Author(s):  
Donald H. Maurice ◽  
Daniel Palmer ◽  
Douglas G. Tilley ◽  
Heather A. Dunkerley ◽  
Stuart J. Netherton ◽  
...  
Keyword(s):  
Cardiovascular System ◽  
Cyclic Nucleotide ◽  
Cyclic Nucleotide Phosphodiesterase ◽  
Phosphodiesterase Activity ◽  
In Cells

scholarly journals Rapid desensitization of vasopressin-stimulated phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine hydrolysis questions the role of these pathways in sustained diacylglycerol formation in A10 vascular-smooth-muscle cells

1992 ◽  
Vol 285 (3) ◽  
pp. 759-766 ◽  
Author(s):  
R Plevin ◽  
M J O Wakelam
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Receptor Antagonist ◽  
Vascular Smooth Muscle Cells ◽  
Inositol Phosphates ◽  
Muscle Cells ◽  
Receptor Activation ◽  
V1a Receptor ◽  
In Cells

The kinetics of vasopressin-stimulated PtdIns(4,5)P2 and phosphatidylcholine (PtdCho) hydrolysis in relation to sustained diacylglycerol (DAG) formation was investigated in A10 vascular-smooth-muscle cells in culture. Vasopressin stimulated a transient increase in Ins(1,4,5)P3 mass formation, which was mirrored by a decrease in PtdIns(4,5)P2 mass levels. Vasopressin stimulated sustained accumulation of total [3H]inositol phosphates ([3H]IP) in the presence of Li+; however, this was significantly decreased by adding a vasopressin-receptor antagonist at different times after initial stimulation. Vasopressin-stimulated phospholipase D (PLD) activity was found to be a transient phenomenon lasting approx. 2 min. Experiments involving agonist preincubation with subsequent addition of butanol confirmed that vasopressin-stimulated PLD activity was desensitized. Vasopressin stimulated an increase in formation of choline, but not of phosphocholine, suggesting that PLD was the major catalytic route of PtdCho hydrolysis in this cell line. The roles of choline and inositol phospholipid hydrolysis in the prolonged phase of DAG formation was examined by comparing vasopressin-stimulated changes in DAG levels in the presence of butanol, the protein kinase C inhibitor Ro-31-8220 or a V1a-receptor antagonist. Vasopressin-stimulated DAG formation was decreased by 40-50% in the presence of butanol between 1 and 10 min; however, during more prolonged stimulation butanol was without significant effect. In cells pretreated with Ro-31-8220, vasopressin-stimulated DAG formation was decreased by approx. 30% at 2 min, but was significantly potentiated at later times. This coincided with an enhancement of vasopressin-stimulated [3H]IP accumulation. In cells exposed to the V1a-receptor antagonist 5 min after addition of vasopressin, subsequent DAG formation was significantly decreased, indicating that sustained formation of DAG, like [3H]IP accumulation, was dependent on continual agonist receptor activation. The results are discussed in terms of different phospholipid-hydrolytic pathways providing DAG generation.

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