BACK TO DIAGRAM CFTR REVIEW PAGE
FOURTH EXTRCELLULAR LOOP
LWLLGNTPLQDKGNSTHSRNNSYAVIITSTS
(amino acids 881 thru 911)
ECL4 is the only extracellular loop besides ECL1 that has been studied in detail and shown to have some function other than simply joining the transmembrane helices. ECL4 is the only place on the structure of CFTR where glycosylation is known to take place. N-glycosylated takes place at two sites on ECL4 (N894 and N900), and this glycosylation is known to require movement from the ER to the Golgi apparatus. Glycosylation is not found at all in deltaF508 mutant CFTR. Functional studies of mutants lacking the N-glycosylation sites reveal that glycosylation is not necessary for normal channel activity or proper folding of CFTR, however some studies show longer I- efflux compared to wild-type activity. It is because of glycosylation that CFTR can exist in three different molecular weight forms with apparent weights of approximately 127, 131, and 160 kDa, representing either nonglycosylated, core glycosylated, or fully mature, complex glycosylated CFTR, respectively, on an SDS-PAGE gel. The FLAG epitope has recently been engineered into this loop, but it had no effect on channel function.
Recent Evidence: More Pieces of the Puzzle?
In November, 2000 O'Riordan, et al (from Genzyme Corporation) took a close look at the carbohydrates on CFTR. They expressed and purified CFTR from both mammalian Chinese hamster ovary (CHO) and insect Sf9 cells and used a combination of techniques involving enzyme glycosidases and FACE analysis (fluorophore-assisted carbohydrate electrophoresis) and found that purified CFTR from CHO cells contained polylactosaminoglycan sequences, while Sf9-CFTR had only oligomannosidic saccharides with fucosylation on the innermost GlcNAc. Polylactosaminoglycans are characteristic of mammalian additions and can be found in T84 cells as well.
In May, 2001, Hammerle et al. reported on a study involving
extracellular loop contributions to both maturation and chloride
activity. They stated that "The first and fourth
extracytoplasmic loops (ELs) contain approximately 15 and 30 residues,
respectively; the other four ELs are extremely short. To examine
the influence of missense mutants in ELs detected in patients with cystic
fibrosis, we have expressed them in mammalian (baby hamster kidney (BHK21))
cells and assessed their biosynthetic processing and chloride channel activity.
In contrast to previous findings that 18 of 30 disease-associated
missense mutations in cytoplasmic loops caused retention of the nascent
polypeptides in the endoplasmic reticulum, all the EL mutants studied matured
and were transported to the cell surface. This pronounced asymmetry
is consistent with the notion that endoplasmic reticulum quality control of
nascent CFTR is exerted primarily on the cytoplasmic side of the membrane.
Although this set of EL mutations has little effect on CFTR maturation, most of
them seriously compromise its chloride channel activity. Substitutions at six
different positions in EL1 and single positions in EL2 and EL4 all destabilized
the open state, some of them severely, indicating that the ELs contribute to the
stability of the CFTR ion pore." J Biol Chem 2001 May
4;276(18):14848-54