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

Abstract

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.

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

“…1B). These results, together with our previous demonstration of both the cell surface and intracellular distribution of endogenous M1-mAChR (Uwada et al, 2011), suggest that the intracellular distribution is specific for the M1 subtype, regardless of its origin. In addition, our data indicate the presence of M1-selective trafficking machinery in N1E-115 neuroblastoma cells.…”

“…Such intracellular localization has also been found for endogenous M1-mAChR in N1E-115 cells (Uwada et al, 2011), and in the cortex and hippocampal neurons of rats, mice and humans (Anisuzzaman et al, 2013;Yamasaki et al, 2010). More recently, we observed constitutive internalization of M1-mAChR that was dependent on a WxxW motif in exogenously transfected HEK293 and HeLa cells (our unpublished observations).…”

“…However, the density of endogenous mAChRs in N1E-115 cells is extremely low in contrast to the brain (,100 versus 3000 fmol/mg protein) (Anisuzzaman et al, 2013;Uwada et al, 2011). Therefore, we transfected exogenous mAChRs in order to more readily analyze receptor dynamics.…”

“…Thus, it has been suggested that there is an M1-subtype-specific mechanism for its intracellular localization. Additionally, we recently reported that intracellular M1-mAChRs are physiologically functional receptors whose activation leads to upregulation of mitogen-activated protein kinase (MAPK) activity and contributes to regulation of synaptic plasticity in hippocampal neurons (Anisuzzaman et al, 2013;Uwada et al, 2011). Therefore, the intracellular localization of M1-mAChR is important for not only the downregulation of signals from cell surface receptors, but also for the upregulation of functional intracellular receptors.…”

Intracrine endothelin signaling evokes IP3-dependent increases in nucleoplasmic Ca2+ in adult cardiac myocytes

“…Similarly, cell permeable and cell impermeable antagonists were recently used to demonstrate that muscarinic and α 1 -adrenergic receptors play distinct roles in function of their localization [97,98]. Considering the number of GPCRs, and their effectors, now known to localize to the nuclear membrane, the biological response evoked upon activation of a given GPCR may be a result of the integration of signalling pathways activated at the plasma membrane and the nuclear membrane.…”