Reconstitution of functional muscarinic receptors by co-expression of amino- and carboxyl-terminal receptor fragments (original) (raw)
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Binding characteristics of the muscarinic receptor subtype of the NG108-15 cell line
Naunyn-schmiedebergs Archives of Pharmacology, 1989
Kinetic, saturation and competition binding studies were conducted on the muscarinic receptor binding sites labeled by [3H]N-methylscopolamine ([3H]NMS) in membranes prepared from NG108-15 cells. The pharmacology of the NG108-15 cell muscarinic receptors was compared to that of the M1 receptors of rat cortex labeled using [3H]pirenzepine, the M2 and M3 receptors of rat heart and submaxillary gland, respectively, labeled using [3H]NMS and the muscarinic receptors of the PC12 cell line also labeled using [3H]NMS. The rate of dissociation of [3H]NMS from the NG10815 cell muscarinic receptor was similar to that obtained at the M3 receptor and at the muscarinic receptor of the P12 cells but was slower that the dissociation rate obtained at the M2 cardiac muscarinic receptor. The Kd of [3H]NMS in the NG108-15 cells was significantly lower than that obtained at the M2 and M3 receptor but was similar to the Kd obtained in PC12 cells. In competition studies the affinity estimates for AF-DX 116, 4-DAMP, methoctramine and pirenzepine were not consistent with the presence of either an M1, M2 Or M3 receptor but were identical to the affinity estimates obtained at the muscarinic receptor of the PC12 cell line. On the basis of these data we conclude that the muscarinic receptor present in the NG108-15 cells is different to the M1, M2 or M3 subtypes already described but is similar to the muscarinic receptor present in the PC12 cell line. Since NG108-15 cells expresses mRNA for the m4 muscarinic receptor gene described by Bonner et al. (1987) we propose that the muscarinic receptors present in this cell line be denoted as M4 receptors.
Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors
Scientific Reports
Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M 2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.
British Journal of Pharmacology, 1995
1 Phosphoinositidase C-linked m3-muscarinic receptors expressed in Chinese hamster ovary cells (CHO-m3 cells) are phosphorylated on serine following agonist stimulation. 2 m3-Muscarinic receptor phosphorylation is concentration-dependent requiring a carbachol concentration of 13.2 /M for half maximal stimulation. 3 The phosphorylation concentration-response curve lies to the left of the curve for carbachol binding to muscarinic receptors (KD = 100 gIM) in membranes from CHO-m3 cells. In contrast, receptor phosphorylation closely correlates with receptor-mediated phosphoinositidase C activation (EC50 for inositol 1,4,5 trisphosphate accumulation during the peak and plateau phases were 7.14 ,M and 5.92 ,UM respectively) but not with rapid agonist-mediated calcium elevation (EC5o = 0.32 gM) measured in fura-2-AM loaded cells. 4 These data suggest a dissociation of receptor phosphorylation from agonist occupation. Such an apparent 'receptor reserve' for m3-muscarinic receptor phosphorylation may be indicative of a mechanism that is dependent on a small amplification of the receptor signal, though probably dissociated from the calcium signal.
Muscarinic Receptors—Characterization, coupling and function
Pharmacology & Therapeutics, 1993
At least five muscarinic receptor genes have been cloned and expressed. Muscarinic receptors act via activation of G proteins: ml, m3 and m5 muscarinic receptors couple to stimulate phospholipase C, while m2 and m4 muscarinic receptors inhibit adenylyl cyclase. This review describes the localization, pharmacology and function of the five muscarinic receptor subtypes. The actions of muscarinic receptors on the heart, smooth muscle, glands and on neurons (both presynaptic and postsynaptic) in the autonomic nervous system and the central nervous system are analyzed in terms of subtypes, biochemical mechanisms and effects on ion channels, including K + channels and Ca 2+ channels. References
Muscarinic M3 receptor coupling and regulation
Life Sciences, 1997
Current concepts regarding the regulation and coupling of muscarinic m3 receptors to G-proteins and various effecters are discussed. The last few years have provided much evidence that although muscarinic ml, m3 and m5 subtypes couple predominantly via pertussis toxin-insensitive G-proteins (Gq/l 1) to activate phosphoinositidase C (PIC), interactions with other G-proteins (Gi, Go, Gs) can be readily observed in cells expressing recombinant muscarinic receptors even at relatively low levels. The significance of this diversity and the potential for agonist "trafficking" could open up opportunities for novel approaches to selective agonist action.
Biomedical Research, 2014
Muscarinic M2, M4, and M2-M4 chimera receptors were transiently expressed in HEK-293 tsA201 cells, and agonist-dependent internalization of these receptors and recycling of internalized receptors were examined by measuring the amount of cell-surface receptors as [ 3 H]N-methylscopolamine (NMS) binding activity. Coexpression of a dominant negative form of dynamin (DNdynamin, dynamin K44A) greatly reduced the agonist-dependent internalization of M4 receptors but not of M2 receptors, as was reported by Vögler et al.
Inhibition of acetylcholine muscarinic M(1) receptor function 2000
1 MT-7 (1 ± 30 nM), a peptide toxin isolated from the venom of the green mamba Dendroaspis angusticeps and previously found to bind selectively to the muscarinic M 1 receptor, inhibited the acetylcholine (ACh)-stimulated [ 35 S]-guanosine-5'-O-(3-thio)triphosphate ([ 35 S]-GTPgS) binding to membranes of Chinese hamster ovary (CHO) cells stably expressing the cloned human muscarinic M 1 receptor subtype. 2 MT-7 failed to aect the ACh-stimulated [ 35 S]-GTPgS binding in membranes of CHO cells expressing either the M 2 , M 3 or M 4 receptor subtype. 3 In N1E-115 neuroblastoma cells endogenously expressing the M 1 and M 4 receptor subtypes, MT-7 (0.3 ± 3.0 nM) inhibited the carbachol (CCh)-stimulated inositol phosphates accumulation, but failed to aect the CCh-induced inhibition of pituitary adenylate cyclase activating polypeptide (PACAP) 38-stimulated cyclic AMP accumulation. 4 In both CHO/M 1 and N1E-115 cells the MT-7 inhibition consisted in a decrease of the maximal agonist eect with minimal changes in the agonist EC 50 value. 5 In CHO/M 1 cell membranes, MT-7 (0.05 ± 25 nM) reduced the speci®c binding of 0.05, 1.0 and 15 nM [ 3 H]-N-methylscopolamine ([ 3 H]-NMS) in a concentration-dependent manner, but failed to cause a complete displacement of the radioligand. Moreover, MT-7 (3 nM) decreased the dissociation rate of [ 3 H]-NMS by about 5 fold.
Biotechnology Letters, 2006
cDNAs encoding for five mAChR subtypes (M1–M5) were cloned under different promoters in various eukaryotic vectors and each subtype was expressed in different mammalian cell lines. CHO-K1 cell line was the best for generating stable cell lines expressing muscarinic receptors. Immunofluorescence and flow cytometry revealed that expression of M1–M5 was primarily localized on the cell membrane. Western blotting and radio-ligand binding studies revealed that expression of each receptor was stable at higher passages.
Molecular analysis of the regulation of muscarinic receptor expression and function
Life Sciences, 1999
We have investigated the molecular mechanisms involved in the regulation of muscarinic acetylcholine receptor gene expression and localization and generated knockout mice to study the role of the M1 muscarinic receptor in vivo. We have used the MDCK cell system to demonstrate that different subtypes of mAChR can be targeted to different regions of polarized cells. We have also examined