Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent Cl- channel at the plasma membrane, and its malfunction results in cystic fibrosis, the most common lethal genetic disease in Caucasians. Quality control of CFTR is strictly regulated by several molecular chaperones. Here we show that calreticulin (CRT), which is a lectin-like chaperone in the endoplasmic reticulum (ER), negatively regulates the cell surface CFTR. RNA interference-based CRT knockdown induced the increase of CFTR expression. Consistently, this effect was observed in vivo. CRT heterozygous (CRT+/-) mice had a higher endogenous expression of CFTR than the wild-type mice. Moreover, CRT overexpression induced cell surface expression of CRT, and it significantly decreased the cell surface expression and function of CFTR. CRT overexpression destabilized the cell surface CFTR by enhancing endocytosis, leading to proteasomal degradation. Deletion of the carboxyl domain of CRT, which results in its ER export, increased the negative effect and enhanced the interaction with CFTR. Thus, CRT in the post-ER compartments may act as a negative regulator of the cell surface CFTR.
"Because CFTR folding takes place in three different compartments, the ER lumen, the ER membrane, and the cytosol, CFTR interacts with several large cellular chaperone and co-chaperone networks (at least 31 components) at various stages of folding (Skach, 2006; Wang et al., 2006). Major chaperone families include cytosolic Hsp70, Hsp90, and their co-chaperones (Yang et al., 1993; Loo et al., 1998; Meacham et al., 1999; Younger et al., 2004; Grove et al., 2011), as well as ER lumenal lectins calnexin and possibly calreticulin (Pind et al., 1994; Harada et al., 2006). "
[Show abstract][Hide abstract] ABSTRACT: In the past decade much has been learned about how Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) folds and misfolds as the etiologic cause of cystic fibrosis (CF). CFTR folding is complex and hierarchical, takes place in multiple cellular compartments and physical environments, and involves several large networks of folding machineries. Insertion of transmembrane (TM) segments into the endoplasmic reticulum (ER) membrane and tertiary folding of cytosolic domains begin cotranslationally as the nascent polypeptide emerges from the ribosome, whereas posttranslational folding establishes critical domain-domain contacts needed to form a physiologically stable structure. Within the membrane, N- and C-terminal TM helices are sorted into bundles that project from the cytosol to form docking sites for nucleotide binding domains, NBD1 and NBD2, which in turn form a sandwich dimer for ATP binding. While tertiary folding is required for domain assembly, proper domain assembly also reciprocally affects folding of individual domains analogous to a jig-saw puzzle wherein the structure of each interlocking piece influences its neighbors. Superimposed on this process is an elaborate proteostatic network of cellular chaperones and folding machineries that facilitate the timing and coordination of specific folding steps in and across the ER membrane. While the details of this process require further refinement, we finally have a useful framework to understand key folding defect(s) caused by ΔF508 that provides a molecular target(s) for the next generation of CFTR small molecule correctors aimed at the specific defect present in the majority of CF patients.
Frontiers in Pharmacology 12/2012; 3:201. DOI:10.3389/fphar.2012.00201 · 3.80 Impact Factor
"These proteins recognize terminal oligosaccharides on proteins modified with high mannose N-linked glycosylation and promote ER retention of “folding intermediates” until they either fold properly or undergo ERAD. As such, Harada et al. (2006, 2007) found that CFTR expression and function were enhanced by RNAi-mediated depletion of calreticulin in both cultured cells and mouse models, suggesting that calreticulin negatively regulates CFTR. Because previous reports indicated that curcumin, a SERCA pump inhibitor, corrected ΔF508-CFTR trafficking to the apical plasma membrane (Egan et al., 2004), Harada et al. (2007) examined the mechanism by which this occurs. "
[Show abstract][Hide abstract] ABSTRACT: Cystic Fibrosis (CF) is the most common autosomal recessive lethal disorder among Caucasian populations. CF results from mutations and resulting dysfunction of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). CFTR is a cyclic AMP-dependent chloride channel that is localized to the apical membrane in epithelial cells where it plays a key role in salt and water homeostasis. An intricate network of molecular chaperone proteins regulates CFTR's proper maturation and trafficking to the apical membrane. Understanding and manipulation of this network may lead to therapeutics for CF in cases where mutant CFTR has aberrant trafficking.
Frontiers in Pharmacology 07/2012; 3:137. DOI:10.3389/fphar.2012.00137 · 3.80 Impact Factor
"In order to silence human CRT expression, the cells were cultured onto 6-well plates, and transfected with CRT siRNA (25 nM, Qiagen, target sequence from ) or negative control siRNA (5′-AATTCTCC- GAACGTGTCACGT-3′, Qiagen) using Lipofectamine™2000 (Invitrogen) according to the manufacturer's instructions. The cells were split after 3–4 days and retransfected. "
[Show abstract][Hide abstract] ABSTRACT: Wnt signalling pathway is a multicomponent cascade involving interaction of several proteins and found to be important for development and function of various cells and tissues. There is increasing evidence that the Wnt/beta-catenin pathway constitutes also one of the essential molecular mechanisms controlling the metabolic aspects of osteoblastic cells. However, in bone, glucocorticoids (GCs) have been reported to weaken Wnt signalling. Therefore, the aim of this study was to characterize the mechanisms behind the cross-talk of these two signalling pathways in human osteoblastic cells. Based on our findings, liganded glucocorticoid receptor (GR) modulated Wnt signalling pathway by decreasing beta-catenin's nuclear accumulation and increasing its relocalization to cell membranes rather than affecting its degradation in human osteoblastic cells. The region of GR responsible for this inhibitory effect located into an area, which harbours the DNA binding as well as nuclear export domains. In further studies, a chaperone protein calreticulin (CRT), known to bind the DNA binding domain of GR and regulate receptor export, was found to be involved in the GR-mediated downregulation of Wnt signalling: GR mutants containing incomplete CRT binding sites were not able to translocate beta-catenin to cell surface. In addition, the inhibitory effect of GCs on endogenous Wnt target gene, cyclin D1, was abolished, when the expression of CRT was attenuated by the RNAi technique. Furthermore, GR and beta-catenin were shown to exist in the same immunocomplex, while interaction between CRT and beta-catenin was observed only in the presence of GR as a mediator molecule. In addition, the GR mutant lacking CRT binding ability impaired the complex formation between beta-catenin and CRT. Together with GR, beta-catenin could thus be co-transported from the nucleus in a CRT-dependent way. These observations represent a novel mechanism for GCs to downregulate Wnt signalling pathway in human osteoblastic cells. Knowledge of these molecular mechanisms is important for understanding the network of multiple signalling cascades in bone environment. Functional Wnt signalling pathway is a prerequisite for proper osteoblastogenesis, and this modulative cross-talk between the steroid pathway and Wnt cascade could therefore explain some of the two-edged effects of GCs on osteoblastic differentiation and function.
Bone 04/2009; 44(4):555-65. DOI:10.1016/j.bone.2008.11.013 · 3.97 Impact Factor
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