Activation of Guanosine 5′-[γ-35S]thio-triphosphate Binding through M1 Muscarinic Receptors in Transfected Chinese Hamster Ovary Cell Membranes: 1. Mathematical Analysis of Catalytic G Protein Activation

2001 ◽  
Vol 59 (4) ◽  
pp. 875-885 ◽  
Author(s):  
Magali Waelbroeck
Keyword(s):  
G Protein ◽  
Muscarinic Receptors ◽  
Mathematical Analysis ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Protein Activation ◽  
G Protein Activation ◽  
M1 Muscarinic ◽  
M1 Muscarinic Receptors

Mutant M1 muscarinic receptors with increased agonist affinity and G protein coupling

Life Sciences ◽  
1999 ◽  
Vol 64 (6-7) ◽  
pp. 563
Author(s):  
W.S. Messer ◽  
X.-P. Huang ◽  
P.I. Nagy ◽  
F.E. Williams ◽  
S.M. Peseckis
Keyword(s):  
G Protein ◽  
Muscarinic Receptors ◽  
G Protein Coupling ◽  
Protein Coupling ◽  
Agonist Affinity ◽  
M1 Muscarinic ◽  
M1 Muscarinic Receptors

Sodium Nitroprusside Induces Internalization of Muscarinic Receptors Stably Expressed in Chinese Hamster Ovary Cell Lines

2002 ◽  
Vol 65 (2) ◽  
pp. 943-946 ◽  
Author(s):  
Roberto Maggio ◽  
Pascaline Barbier ◽  
Andrea Toso ◽  
Davide Barletta ◽  
Giovanni U. Corsini
Keyword(s):  
Sodium Nitroprusside ◽  
Cell Lines ◽  
Chinese Hamster Ovary Cell ◽  
Muscarinic Receptors ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Ovary Cell

scholarly journals Agonist-induced desensitization and phosphorylation of m1-muscarinic receptors

1999 ◽  
Vol 338 (1) ◽  
pp. 175-183 ◽  
Author(s):  
Mark G. WAUGH ◽  
R. A. John CHALLISS ◽  
Gabriel BERSTEIN ◽  
Stefan R. NAHORSKI ◽  
Andrew B. TOBIN
Keyword(s):  
Muscarinic Receptor ◽  
Muscarinic Receptors ◽  
Chinese Hamster ◽  
Receptor Internalization ◽  
Receptor Subtype ◽  
Muscarinic Agonist ◽  
Dependent Manner ◽  
Receptor Desensitization ◽  
M1 Muscarinic ◽  
M1 Muscarinic Receptors

Pre-stimulation of Chinese hamster ovary (CHO) cells expressing the human m1-muscarinic receptor (CHO-m1 cells) with a maximally effective concentration of the muscarinic agonist methacholine resulted in desensitization of Ins(1,4,5)P3 accumulation, apparent as a ∼ 4-fold shift in the agonist dose–response curve. Agonist-induced desensitization was rapid (detectable by 10 s) and concentration dependent (EC50 = 8.2±2.2 µM) and resulted in a complete loss of receptor reserve for the agonist-stimulated Ins(1,4,5)P3 response. An investigation of the possible mechanisms involved in m1-muscarinic receptor desensitization indicated that agonist-induced receptor internalization, PtdIns-(4,5)P2 depletion or an increased rate of Ins(1,4,5)P3 metabolism were not involved. m1-Muscarinic receptors did, however, undergo rapid agonist-induced phosphorylation with a time course that was consistent with an involvement in receptor desensitization. Characterization studies indicated that the receptor-specific kinase involved was distinct from protein kinase C and other second-messenger-dependent protein kinases. Since previous studies have suggested that the m3-muscarinic receptor subtype undergoes agonist-dependent phosphorylation via casein kinase 1α (CK1α) [Tobin, Totty, Sterlin and Nahorski (1997) J. Biol. Chem. 272, 20844–20849], we examined the ability of m1-muscarinic receptors to be phosphorylated by this kinase. In reconstitution experiments, CK1α was able to phosphorylate purified, soluble m1-muscarinic receptors in an agonist-dependent manner.

Development of Flashplate™ Technology to Measure [35S]GTPyS Binding to Chinese Hamster Ovary Cell Membranes Expressing the Cloned Human 5-HTIB Receptor

1998 ◽  
Vol 3 (2) ◽  
pp. 101-105 ◽  
Author(s):  
J. Watson ◽  
J. V. Selkirk ◽  
A. M. Brown
Keyword(s):  
G Protein ◽  
Chinese Hamster Ovary ◽  
Radioligand Binding ◽  
Partial Agonist ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Intrinsic Activity ◽  
Filtration Method ◽  
Gtpγs Binding

With the exponential rate at which proposed drugs are being produced for disease therapy, it is now essential to automate assays used to screen these compounds and increase throughput. This has been rapidly adopted for simple radioligand binding assays but is less amenable for certain functional screens. [35S]GTPγS binding represents a convenient method for screening ligands that bind to G protein-coupled receptors and, ultimately, stimulate G-protein activation. In this study we have investigated the use of 96-well FlashPlates™ (NEN DuPont, Stevenage, England) to measure [35S]GTPγS binding to human 5-HT1B receptors expressed in Chinese hamster ovary cells. The cells were added to the individual wells of the FlashPlate and incubated with [35S]GTPγS in the presence or absence of test drug and bound radioactivity measured in a 96-well spectrometer. 5-HT produced a stimulation of basal [35S]GTPγS binding, which was robust within and between experiments, with pEC50 = 8.1. The 5-HT1B partial agonist GR127935 (2′n-methyl-4′-5-methyl-1,2,4 oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide) caused a partial stimulation (pEC5o = 8.3, intrinsic activity = 0.7), and the selective 5-HTIB receptor antagonist SB-224289 (2,3,6,7-tetrahydro-1′-methyl-5-{2′-methyl-4′-[(5-methyl-1,2,4-oxadiazole-3-yl)biphenyl-4-yl]carbonyl}furo[2,3-flindole-3-spiro-4′-piperidine oxalate) displayed inverse agonism with pEC50 = 7.6. These results are similar to those obtained using the conventional filtration method and indicate that FlashPlate technology can provide a rapid method for measuring [35S]GTPγS binding.

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