ABCMdb

PMID: 16339147

Jurkuvenaite A, Varga K, Nowotarski K, Kirk KL, Sorscher EJ, Li Y, Clancy JP, Bebok Z, Collawn JF
Mutations in the amino terminus of the cystic fibrosis transmembrane conductance regulator enhance endocytosis.
J Biol Chem. 2006 Feb 10;281(6):3329-34. Epub 2005 Dec 8., 2006-02-10 [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu In the present studies, two naturally occurring cystic fibrosis mutations in the amino terminus of CFTR, R31C, and R31L were examined. Login to comment 8 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu Together, the results suggest that both R31C and R31L mutations compromise biogenesis and enhance internalization of CFTR. Login to comment 25 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu In the present studies, we examined two naturally occurring mutations, R31C and R31L, which cause mild CF.3 To determine the defects caused by these missense mutations, we expressed them in COS-7 cells and analyzed their biogenesis and trafficking. Login to comment 26 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu Our results indicate that both R31C and R31L have compromised biogenesis and enhanced endocytosis compared with wild-type CFTR. Login to comment 29 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu MATERIALS AND METHODS Construction of CFTR Mutants-The R31C and R31L mutants were prepared by PCR mutagenesis. Login to comment 32 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu Using the CFTR gene contained in plasmid pCDNA3.1ϩ as template, the R31L and R31C mutants were constructed by PCR-mutagenesis using a pair of internal primers for each mutant, annealing at the Arg-31 coding site, and a pair of external primers, one annealing at the Nhe-I site of the multilinker of the vector, and one annealing at the BspE-I site in the CFTR gene. Login to comment 96 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu EC, extracellular; IC, intracellular; L/C, R31L and R31C mutations. Login to comment 102 ABCC7 p.Arg31Cys ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu ABCC7 p.Arg31Leu With the R31C and R31L mutants, however, very little processing occurredduringthefirst2hofchase(0and4.7 Ϯ 1.5%,respectively).By4h, 10.3 Ϯ 3.0% (R31C) and 11.3 Ϯ 2.1% (R31L) of CFTR was converted to the mature form, indicating that, although there is a processing defect, it does not cause complete inhibition. Login to comment 106 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu The results indicate that the protein half-life of the wild-type protein is 13.3 Ϯ 1.2 h, and the R31C and R31L half-lives are 12.7 Ϯ 0.6 and 14.3 Ϯ 3.1 h, respectively, indicating that, FIGURE 3. Login to comment 108 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu Wild-type, R31C, and R31L CFTR half-lives were determined in COS-7 cells 24 h after transfection. Login to comment 111 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu A, representative gels of wild-type (WT), R31C, and R31L CFTR half-lives are shown. Login to comment 115 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu Protein maturation of R31C and R31L CFTR is inefficient compared with wild-type CFTR. Login to comment 120 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu A, representative metabolic labeling experiments are shown for wild type (WT), R31C, and R31L (left panels). Login to comment 126 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu Maturation efficiencies of wild type, R31C, and R31L were calculated after 4 h of chase (average Ϯ S.D., n ϭ 3; *, p Ͻ 0.005; **, p Ͻ 0.001). Login to comment 127 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu C, R31C (C) and R31L (L) are not temperature-sensitive. Login to comment 128 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu WT, R31C and R31L CFTR were immunoprecipitated from COS-7 cells 48 h after transfection. Login to comment 131 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu A 27 °C incubation increased the amount of B band but did not influence C band production for R31C or R31L CFTR. Login to comment 133 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu Reduced Surface Expression of R31C and R31L-Because some mutant protein was processed correctly and the protein half-life appeared normal, the surface pool of the Arg-31 mutants was examined next. Login to comment 136 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu For the R31C and R31L mutants, however, staining was much more restricted to an intracellular, reticular pattern. Login to comment 138 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu To confirm this observation, cells expressing wild-type, R31C, and R31L CFTR were surface-biotinylated (Fig. 4B), CFTR was immunoprecipitated, and the biotinylated fraction was detected by Western blot analysis. Login to comment 139 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu The results indicate that the surface pool of the R31C and R31L mutants is 20.3 Ϯ 7.5 and 33.6 Ϯ 11.7% of the wild-type protein, respectively (n ϭ 4, p Ͻ 0.005, and p Ͻ 0.05, respectively. Login to comment 141 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu R31C and R31L Are Internalized More Rapidly than the Wild-type CFTR-Because the surface pool was smaller than predicted, we next tested whether the mutations affected CFTR clearance from the cell surface. Login to comment 145 ABCC7 p.Arg31Cys ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu ABCC7 p.Arg31Leu The R31C and R31L mutants, however, have dramatically altered internalization kinetics with 54 Ϯ 4 and 55 Ϯ 13.9% of the R31C and R31L mutants, respectively (internalized during the same warm-up period). Login to comment 147 ABCC7 p.Arg31Cys ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu ABCC7 p.Arg31Leu The Functional Activity of the R31C and R31L Mutants Is Severely Compromised-As a final measure of the total CFTR chloride channels at the cell surface, we tested the functional activity of the R31C and R31L mutants in two complementary functional assays, macroscopic patch clamp experiments and SPQ assays. Login to comment 152 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu Cells transfected with the R31L and R31C mutants exhibited two differences as compared with wild-type-transfected cells. Login to comment 157 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu R31C and R31L surface expression is lower than wild-type CFTR. Login to comment 158 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu A, wild-type, R31C, and R31L CFTR distributions were examined in COS-7 cells 48 h after transfection using indirect immunofluorescence. Login to comment 160 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu Arrows indicate the prominant cell surface expression of the wild-type protein CFTR and the diminished amount of surface staining in the R31C and R31L CFTR-expressing cells. Login to comment 165 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu Internalization of the R31C and R31L CFTR mutants is dramatically enhanced compared with the wild-type protein. Login to comment 166 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu A, wild-type, R31C, and R31L CFTR internalization in COS-7 cells. Login to comment 181 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu ABCC7 p.Arg31Leu DISCUSSION The R31C and R31L are naturally occurring missense CF mutations that appear to have a mild phenotype (31).3 Both of these mutations are rare (identified once in 284 CF chromosomes, for the R31L mutation). Login to comment 182 ABCC7 p.Arg31Cys The R31C mutation was found in a 45-year-old male CF patient diagnosed in childhood who was pancreatic-sufficient with moderate pulmonary symptoms and a positive sweat test. Login to comment 183 ABCC7 p.Ile556Val His other allele is the I556V mutation. Login to comment 184 ABCC7 p.Arg31Leu The R31L was found in a 24-year-old female CF patient who was pancreatic-sufficient with normal lung function but a positive sweat test. Login to comment 194 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu R31C and R31L mutants have diminished channel activity compared with the wild-type protein. Login to comment 195 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu A-C, representative current traces for wild-type (WT) and R31C and R31L mutants. Login to comment 204 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu Note that the data for the L and C mutants overestimate their functional activities, because unlike WT, most excised mutant patches exhibited undetectable CFTR activity and were excluded from this analysis (R31L, 4 active patches of 13 total; R31C, 4 active patches of 19 total; wild type, 4 active patches of 6 total). Login to comment 206 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu E, functional analysis of wild-type and R31C and R31L CFTR using SPQ fluorescence. Login to comment 207 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu The change in SPQ fluorescence is shown for COS-7 cells expressing wild-type, R31C, and R31L CFTR. Login to comment 222 ABCC7 p.Asn287Tyr Another CFTR mutation was recently identified in the second intracellular loop, N287Y, which affects endocytosis over wild-type levels (34). Login to comment 223 ABCC7 p.Asn287Tyr Interestingly, the N287Y mutation does not introduce a consensus tyrosine-based signal and did not affect biogenesis of CFTR. Login to comment 232 ABCC7 p.Arg31Cys ABCC7 p.Asn287Tyr ABCC7 p.Arg31Leu Analysis of the ⌬F508, N287Y, and R31L and R31C indicate that alterations in the transport of CFTR at the cell surface, whether it is enhanced internalization or compromised recycling, can result in a disease phenotype. Login to comment