Different affinities of native α1B-adrenoceptors for ketanserin between intact tissue segments and membrane preparations

Sathi, Zakia Sultana; Anisuzzaman, Abu Syed Md; Morishima, Shigeru; Suzuki, Fumiko; Tanaka, Takashi; Yoshiki, Hatsumi; Muramatsu, Ikunobu

doi:10.1016/j.ejphar.2008.02.003

Cited by 15 publications

(7 citation statements)

References 29 publications

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“…N1E‐115 cells were harvested by gentle scraping without trypsinization. Whole‐cell binding assays were performed as previously described (Sathi et al. 2008) in Krebs‐HEPES buffer (140 mM NaCl, 5.4 mM KCl, 1.2 mM MgCl 2 , 2.0 mM CaCl 2 , 0.3 mM NaH 2 PO 4 , 11.1 mM glucose, 5 mM HEPES, pH 7.4) in a final volume of 1 mL for 4 h at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Intracellular distribution of functional M₁‐muscarinic acetylcholine receptors in N1E‐115 neuroblastoma cells

Uwada¹,

Anisuzzaman²,

Nishimune

et al. 2011

Journal of Neurochemistry

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J. Neurochem. (2011) 118, 958–967. Abstract Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E‐115 neuroblastoma cells, we detected both plasma membrane and intracellular M1‐mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M1‐mAChRs were detected by the hydrophobic ligand probe, 1‐quinuclidinyl‐[phenyl‐4‐3H]‐benzilate ([3H]‐QNB) whereas the hydrophilic probe, 1‐[N‐methyl‐3H] scopolamine ([3H]‐NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M1‐mAChR antibody also detected both cell‐surface and intracellular M1‐mAChRs. Carbachol‐stimulated phosphatidylinositol hydrolysis and Ca2+ mobilization were completely inhibited by a cell‐impermeable M1 antagonist, muscarinic toxin ‐7 and the Gq/11 inhibitor YM‐254890. However, carbachol‐stimulated extracellular‐regulated kinase 1/2 activation was unaffected by muscarinic toxin‐7, but was blocked by the cell‐permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of Gq/11 (YM‐254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M1‐mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell‐impermeable versus cell‐permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes.

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Section: Methodsmentioning

confidence: 99%

Intracellular distribution of functional M₁‐muscarinic acetylcholine receptors in N1E‐115 neuroblastoma cells

Uwada¹,

Anisuzzaman²,

Nishimune

et al. 2011

Journal of Neurochemistry

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“…After 36 h of culture, the cells were subjected to whole-cell binding assays at 4˚C as described previously (Sathi et al, 2008).…”

Section: Immunofluorescence Confocal Microscopymentioning

confidence: 99%

Intracellular localization of M1 muscarinic acetylcholine receptor through clathrin-dependent constitutive internalization via a C-terminal tryptophan-based motif

Uwada¹,

Yoshiki²,

Masuoka³

et al. 2014

Journal of Cell Science

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The M1 muscarinic acetylcholine receptor (M1-mAChR, encoded by CHRM1) is a G-protein-coupled membrane receptor that is activated by extracellular cholinergic stimuli. Recent investigations have revealed the intracellular localization of M1-mAChR. In this study, we observed constitutive internalization of M1-mAChR in mouse neuroblastoma N1E-115 cells without agonist stimulation. Constitutive internalization depended on dynamin, clathrin and the adaptor protein-2 (AP-2) complex. A WxxI motif in the M1-mAChR C-terminus is essential for its constitutive internalization, given that replacement of W 442 or I 445 with alanine residues abolished constitutive internalization. This WxxI motif resembles YxxW, which is the canonical binding motif for the m2 subunit of the AP-2 complex. The M1-mAChR C-terminal WxxI motif interacted with AP-2 m2. W442A and I445A mutants of the M1-mAChR C-terminal sequence lost AP-2-m2-binding activity, whereas the W442Y mutant bound more effectively than wild type. Consistent with these results, W442A and I445A M1-mAChR mutants selectively localized to the cell surface. By contrast, the W442Y receptor mutant was found only at intracellular sites. Our data indicate that the cellular distribution of M1-mAChR is governed by the C-terminal tryptophanbased motif, which mediates constitutive internalization.

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“…A binding assay that uses intact tissue segments has been shown to be a powerful method for analysis of the intrinsic properties of receptors present in native tissues (Muramatsu et al, 2005Anisuzzaman et al, 2008a;Sathi et al, 2008;Su et al, 2008). When using the intact segment binding assay, in contrast to conventional membrane binding assays, it is not necessary to consider either the change of receptor characteristics that may occur upon tissue homogenization that eliminates the receptor's natural environment or the selective loss of receptors upon isolating membranes by differential centrifugation (Muramatsu et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

Influence of Tissue Integrity on Pharmacological Phenotypes of Muscarinic Acetylcholine Receptors in the Rat Cerebral Cortex

Anisuzzaman

Nishimune²,

Yoshiki³

et al. 2011

J Pharmacol Exp Ther

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Different affinities of native α1B-adrenoceptors for ketanserin between intact tissue segments and membrane preparations

Cited by 15 publications

References 29 publications

Intracellular distribution of functional M₁‐muscarinic acetylcholine receptors in N1E‐115 neuroblastoma cells

Intracellular distribution of functional M₁‐muscarinic acetylcholine receptors in N1E‐115 neuroblastoma cells

Intracellular localization of M1 muscarinic acetylcholine receptor through clathrin-dependent constitutive internalization via a C-terminal tryptophan-based motif

Influence of Tissue Integrity on Pharmacological Phenotypes of Muscarinic Acetylcholine Receptors in the Rat Cerebral Cortex

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Different affinities of native α1B-adrenoceptors for ketanserin between intact tissue segments and membrane preparations

Cited by 15 publications

References 29 publications

Intracellular distribution of functional M1‐muscarinic acetylcholine receptors in N1E‐115 neuroblastoma cells

Intracellular distribution of functional M1‐muscarinic acetylcholine receptors in N1E‐115 neuroblastoma cells

Intracellular localization of M1 muscarinic acetylcholine receptor through clathrin-dependent constitutive internalization via a C-terminal tryptophan-based motif

Influence of Tissue Integrity on Pharmacological Phenotypes of Muscarinic Acetylcholine Receptors in the Rat Cerebral Cortex

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Intracellular distribution of functional M₁‐muscarinic acetylcholine receptors in N1E‐115 neuroblastoma cells

Intracellular distribution of functional M₁‐muscarinic acetylcholine receptors in N1E‐115 neuroblastoma cells