Calreticulin Negatively Regulates the Cell Surface Expression of Cystic Fibrosis Transmembrane Conductance Regulator

Abstract

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.Cystic fibrosis transmembrane conductance regulator (CFTR) 3 is a polytopic integral membrane protein, which functions as a cAMP-dependent Cl Ϫ channel at the plasma membrane (1-4). Mutations in the CFTR gene lead to the absence or malfunction of a regulated Cl Ϫ channel in the apical membrane of secretory epithelia resulting in the clinical symptoms of cystic fibrosis, which is the most common lethal genetic disease in Caucasians (5). After translation, CFTR is folded in the endoplasmic reticulum (ER) assisted by several chaperone proteins, but only ϳ30% of the immature CFTR is exported from the ER to the Golgi and converted to the mature form by oligosaccharide processing (6). Ultimately, mature CFTR reaches the plasma membrane where it functions as a regulated Cl Ϫ channel (6). However, ϳ70% of immature CFTR is retained in the ER by the ER quality control system (6, 7). The ERretained immature CFTR is ultimately ubiquitinated by an ubiquitin ligase, CHIP (carboxyl terminus of HSC70-interacting protein), and degraded by proteasomes, which is known as ER-associated degradation (ERAD) (8 -11). In contrast, all ⌬F508 CFTR (ϳ 99%), the most common mutant of CFTR in cystic fibrosis patients, is retained in the ER, leading to ERAD (6, 7). Several chaperone proteins are involved in the quality control of CFTR. During insertion into the ER membrane, cytosolic chaperones Hsp70/Hsc70, Hdj-2, and Hsp90 interact with immature CFTR to stimulate productive folding (12-14). Moreover, cytosolic HspBP1 attenuates the activity of CHIP ubiquitin ligase, resulting in the inhibition of ERAD and stimulation of CFTR maturation (15). An ER-resident lectinlike chaperone, calnexin (CNX), also participates in the quality control of CFTR (7, 16). CNX interacts with immature CFTR via monoglucosylated oligosaccharide to attenuate the ERAD, resulting in the stimulation of CFTR folding (7...

ERp29 Regulates ΔF508 and Wild-type Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Trafficking to the Plasma Membrane in Cystic Fibrosis (CF) and Non-CF Epithelial Cells

“…We have seen and extensively discussed potential mechanisms underlying similar effects for chaperone regulation of ENaC trafficking in oocytes (49), and there are other such reports for ER chaperones (15,64).…”

“…Molecular chaperones are potential therapeutic targets to facilitate improvement of ⌬F508-CFTR trafficking, but so far, little is known about the exact role of the chaperones in CFTR folding and trafficking. Of particular note, investigations of classical ER chaperones, including BiP/grp78, endoplasmin/grp94, and calreticulin, have failed to demonstrate a central role for these ER chaperones in promoting the proper folding, assembly, and trafficking of CFTR (2,3,6,15). Sodium 4-phenylbutyrate (4PBA) improves ⌬F508-CFTR intracellular trafficking in CF epithelial cells such as the IB3-1 CF human bronchiolar epithelial cell line (genotype ⌬F508/ W1282X) as early as 4 -8 h after exposure and restores CFTR function at the plasma membrane without altering CFTR mRNA expression (16).…”

Calreticulin chaperones regulate functional expression of vomeronasal type 2 pheromone receptors

“…Calreticulin was shown to promote ER retention of CFTR (35,36), because depletion of calreticulin enhances CFTR to be trafficked to the plasma membrane. In the future it would be interesting to ask whether calreticulin4 could allow proper CFTR trafficking while suppressing the undesirable UPR.…”

Insertion of an Arginine Residue into the Transmembrane Segments Corrects Protein Misfolding

“…The mechanism of how pharmacological chaperones rescue folding defects in P-gp (11) is controversial because it is difficult to distinguish whether increased maturation was due to direct effects on TM packing (12) or due to indirect effects on cellular folding pathways. For example, stress-induced alterations in molecular chaperone levels (13)(14)(15) or endoplasmic reticulum-associated degradation (16) could potentially promote maturation of ⌬F508 CFTR or ⌬Y490 P-gp. Therefore, it is important to demonstrate that manipulation of the TM segments can promote folding of mutant proteins in the absence of potential stress-induced cell responses.…”