Characterization and regulation of the expression of scyllatoxin (Leiurotoxin I) receptors in the human neuroblastoma cell line NB-OK 1

Abstract125I-[Tyr2]scyllatoxin allowed to label a single class of high-affinity receptors in membranes from the human neuroblastoma cell line NB-OK 1. The Kd of these receptors was 60 pM for scyllatoxin (Leiurotoxin I) and 20 pM for apamin and the Bmax was low (3.8 fmol/mg membrane protein). K+ increased toxin binding at low concentrations but exerted opposite effects at high concentrations. Ca2+, guanidinium and Na+ exerted only inhibitory effects on binding. Scyllatoxin binding sites were overexpressed 2.5-fold after a 24-h cell pretreatment with 2 mM butyrate. This effect was suppressed by cycloheximide

Identification of drugs competing with d-tubocurarine for an allosteric site on cardiac muscarinic receptors

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Activation of guanosine 5'-[gamma-(35)S]thio-triphosphate binding through M(1) muscarinic receptors in transfected Chinese hamster ovary cell membranes; 1. Mathematical analysis of catalytic G protein activation.

I analyzed in this work the effect of agonists and unlabeled guanyl nucleotides on [(35)S]GTP gamma S and [(3)H]NMS binding to transfected CHO cells expressing hM(1) muscarinic receptors. I was unable to explain my kinetic results by "traditional" (one-site, two-site, or two-step) bimolecular binding models. I therefore examined the equations that describe catalytic G protein activation. My results were fully consistent with the following interpretation: G protein-coupled receptors either interacted with GDP-bound G proteins and facilitated the GDP release or recognized empty G proteins, depending on the incubation conditions. The receptor-coupled empty G proteins (RG) then recognized GTP gamma S, and the occupied G protein (G) dissociated reversibly from the receptor. Agonists accelerated the GDP release from receptor-coupled G proteins and accelerated the G dissociation: both effects accelerated synergically the G protein-GTP gamma S association reaction in the presence of GDP. GTP gamma S-bound G proteins, G, competed efficiently with inactive (empty or GDP-bound) G proteins for receptor recognition, and were able, therefore, at low concentrations, to quench the [(35)S]GTP gamma S binding reaction.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Allosteric drugs acting at muscarinic acetylcholine receptors.

The binding properties of muscarinic acetylcholine receptors are affected by various drugs acting at a second (allosteric) binding site, usually (but not always) at supratherapeutic concentrations. Allosteric drugs acting at GABA receptors present advantages over competitive drugs; this explains the interest raised by allosteric effects on muscarinic receptors. A theoretical and practicable definition of allosteric drugs acting at muscarinic receptors will be given in this work, together with a summary of recent data concerning the number, position, and structural requirements of their binding sites.Journal ArticleResearch Support, Non-U.S. Gov'tReviewinfo:eu-repo/semantics/publishe

The pH dependence of insulin binding. A quantitative study.

A model is presented to study quantitative the effect of pH on a ligand-receptor interaction. Assuming that binding is only possible if all the "active groups" are in the correct ionic state, and that the ionic state of the other residues does not affect the association constant, it is possible to measure the number and pK values of the active groups. This model is applied to insulin and insulin analogs binding to its cellular receptor. Two active groups are responsible for the marked pH dependence of the reaction: a deprotonated residue of pK 7.6 at 25 degrees C (ionization heat: 1.5 kcal .mol-1) and a protonated residue of pK 8.0 at 25 degrees C (ionization heat: 12 kcal .mol-1). The first active group might be a carboxyl residue, in a hydrophobic environment, probably belonging to the receptor molecule. The second active group was further identified as the A1 alpha-amino residue of insulin by the study of insulin analogs.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Activation of guanosine 5'-[gamma-(35)S]thio-triphosphate binding through M(1) muscarinic receptors in transfected Chinese Hamster ovary cell membranes: 2. Testing the "two-states" model of receptor activation.

I suggested in the accompanying article [Mol Pharmacol 2001;59:875-885] that muscarinic receptors catalyzed G protein activation. Acetylcholine or carbamylcholine recognition facilitated not only the GDP release from receptor-coupled inactive G proteins but also the release of G from the (unstable) HRG complex. The two effects facilitated [(35)S]GTP gamma S binding in the presence of GDP, but could be studied separately by comparing [(35)S]GTP gamma S binding in the absence and presence of GTP. Guanyl nucleotides affected the efficiency of receptor-G protein coupling. The relative efficacies of partial agonists in the absence and presence of GTP should remain nonlinearly correlated if all agonists stabilize (to different extents) the same active receptor conformation. The correlation between M(1) muscarinic agonists' efficacy in accelerating [(35)S]GTP gamma S binding in the absence of other nucleotides and their in vivo efficacy (inositol phosphate accumulation) was in fact very poor. This probably reflected the presence of GTP in intact cells: pertussis toxin pretreatment (which inactivates the G(i/o) proteins) did not affect the agonists' efficacy profile (evaluated in the absence of spare receptors), but the addition of GTP to the [(35)S]GTP gamma S binding medium did. These results did not support the allosteric "two states" model of receptor activation, but suggested that different agonists induced different receptor conformations ("induced fit").Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Heterogeneity of neurotransmitter receptors: A comparison of beta-adrenergic, muscarinic cholinergic, and VIP receptors

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Receptor theory

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Stimulation by inophore A23187 and carbamylcholine of prostaglandins release from the thyroid gland in vitro [proceedings].

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