Functional characterization of the CFTR R domain using CFTR/MDR1 hybrid and deletion constructs.
To improve our insight into the structure and function of the CFTR R domain, deletion and hybrid constructs in which different parts of the R domain were deleted or replaced by the MDR1 linker domain, and vice versa, were made. Replacement of the linker domain by the R domain did not result in a decrease and replacement of the CFTR R domain by the linker domain did not result in an increase of maturation efficiency, when compared to the respective wild-type proteins. This indicates that the R domain is not responsible for the high degree of degradation observed for CFTR translation products in the ER, but rather the overall structure or sequences located outside the R domain. Replacing the C-terminal part of the R domain (amino acids 780-830) by the MDR1 linker domain resulted in the appearance of PKA-dependent whole cell chloride currents which were not significantly different from wild-type CFTR currents. This might indicate that the PKA sites present in the linker domain are functional and that not the exact sequence of the C-terminal part of the R domain is important, but rather the presence of PKA sites and the length. Moreover, when this hybrid construct was PKC-stimulated, chloride currents were activated. Although these PKC-induced currents were lower than the PKA-induced ones, this again indicates that the linker domain is functional in this hybrid construct. Taken together, these results suggest that the MDR1 linker domain can substitute for part of the regulatory domain of the CFTR protein.
Vankeerberghen
A
A
Center for Human Genetics and Laboratory of Physiology, University of Leuven, Gasthuisberg O&N6, Herestraat 49, B-3000 Leuven, Belgium. anne.vankeerberghen@med.kuleuven.ac.be
Lin
W
W
Jaspers
M
M
Cuppens
H
H
Nilius
B
B
Cassiman
J J
JJ
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D018118:Chloride Channels / Q000378:metabolism; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; C505032:CFTR protein, human; D018118:Chloride Channels; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017868:Cyclic AMP-Dependent Protein Kinases