Endocytic sorting and downregulation of the M2 acetylcholine receptor is regulated by ubiquitin and the ESCRT complex.

Cholinergic dysfunction plays a critical role in a number of disease states, and the loss of functional muscarinic acetylcholine receptors plays a key role in disease pathogenesis. Therefore, preventing receptor downregulation would maintain functional receptor number, and be predicted to alleviate symptoms. However, the molecular mechanism(s) underlying muscarinic receptor downregulation are currently unknown. Here we demonstrate that the M2 muscarinic receptor undergoes rapid lysosomal proteolysis, and this lysosomal trafficking is facilitated by ubiquitination of the receptor. Importantly, we show that this trafficking is driven specifically by ESCRT mediated involution. Critically, we provide evidence that disruption of this process leads to a re-routing of the trafficking of the M2 receptor away from the lysosome and into recycling pathway, and eventually back to the plasma membrane. This study is the first to identify the process by which the M2 muscarinic acetylcholine receptor undergoes endocytic sorting, and critically reveals a regulatory checkpoint that represents a target to pharmacologically increase the number of functional muscarinic receptors within the central nervous system.

(Pro)renin receptor contributes to regulation of renal epithelial sodium channel.

BACKGROUND: Recent studies reported increased (Pro)renin receptor (PRR) expression during low-salt intake. We hypothesized that PRR plays a role in regulation of renal epithelial sodium channel (ENaC) through serum and glucocorticoid-inducible kinase isoform 1 (SGK-1)-neural precursor cell expressed, developmentally downregulated 4-2 (Nedd4-2) signaling pathway. METHOD: Male Sprague-Dawley rats on normal-sodium diet and mouse renal inner medullary collecting duct cells treated with NaCl at 130  mmol/l (normal salt), or 63  mmol/l (low salt) were studied. PRR and α-ENaC expressions were evaluated 1 week after right uninephrectomy and left renal interstitial administration of 5% dextrose, scramble shRNA, or PRR shRNA (n = 6 each treatment). RESULTS: In-vivo PRR shRNA significantly reduced expressions of PRR throughout the kidney and α-ENaC subunits in the renal medulla. In inner medullary collecting duct cells, low salt or angiotensin II (Ang II) augmented the mRNA and protein expressions of PRR (P < 0.05), SGK-1 (P < 0.05), and α-ENaC (P < 0.05). Low salt or Ang II increased the phosphorylation of Nedd4-2. In cells treated with low salt or Ang II, PRR siRNA significantly downregulated the mRNA and protein expressions of PRR (P < 0.05), SGK-1 (P < 0.05), and α-ENaC expression (P < 0.05). CONCLUSION: We conclude that PRR contributes to the regulation of α-ENaC via SGK-1-Nedd4-2 signaling pathway.

The phosphorylation of endogenous Nedd4-2 In Na(+)-absorbing human airway epithelial cells.

Neural precursor cell expressed, developmentally down-regulated protein 4-2 (Nedd4-2) mediates the internalisation / degradation of epithelial Na(+) channel subunits (α-, ß- and γ-ENaC). Serum / glucocorticoid inducible kinase 1 (SGK1) and protein kinase A (PKA) both appear to inhibit this process by phosphorylating Nedd4-2-Ser(221), -Ser(327) and -Thr(246). This Nedd4-2 inactivation process is thought to be central to the hormonal control of Na(+) absorption. The present study of H441 human airway epithelial cells therefore explores the effects of SGK1 and / or PKA upon the phosphorylation / abundance of endogenous Nedd4-2; the surface expression of ENaC subunits, and electrogenic Na(+) transport. Effects on Nedd4-2 phosphorylation/abundance and the surface expression of ENaC were monitored by western analysis, whilst Na(+) absorption was quantified electrometrically. Acutely (20min) activating PKA in glucocorticoid-deprived (24h) cells increased the abundance of Ser(221)-phosphorylated, Ser(327)-phosphorylated and total Nedd4-2 without altering the abundance of Thr(246)-phosphorylated Nedd4-2. Activating PKA under these conditions did not cause a co-ordinated increase in the surface abundance of α-, ß- and γ-ENaC and had only a very small effect upon electrogenic Na(+) absorption. Activating PKA (20min) in glucocorticoid-treated (0.2µM dexamethasone, 24h) cells, on the other hand, increased the abundance of Ser(221)-, Ser(327)- and Thr(246)-phosphorylated and total Nedd4-2; increased the surface abundance of α-, ß- and γ-ENaC and evoked a clear stimulation of Na(+) transport. Chronic glucocorticoid stimulation therefore appears to allow cAMP-dependent control of Na(+) absorption by facilitating the effects of PKA upon the Nedd4-2 and ENaC subunits.

The relationship between ER-multivesicular body membrane contacts and the ESCRT machinery.

Activated EGFR (epidermal growth factor receptor) undergoes ESCRT (endosomal sorting complex required for transport)-mediated sorting on to ILVs (intraluminal vesicles) of endosomes before degradation in the lysosome. Sorting of endocytosed EGFR on to ILVs removes the catalytic domain of the EGFR from the cytoplasm, resulting in termination of receptor signalling. EGFR signalling is also subject to down-regulation through receptor dephosphorylation by the ER (endoplasmic reticulum)-localized PTP1B (protein tyrosine phosphatase 1B). PTP1B on the cytoplasmic face of the ER interacts with endocytosed EGFR via direct membrane contacts sites between the ER and endosomes. In the present paper, we review the relationship between ER-endosome membrane contact sites and ILV formation, and their potential role in the regulation of EGFR sorting on to ILVs, through PTP1B-mediated dephosphorylation of both EGFR and components of the ESCRT machinery.