ABCMdb

PMID: 7757078

Smit LS, Strong TV, Wilkinson DJ, Macek M Jr, Mansoura MK, Wood DL, Cole JL, Cutting GR, Cohn JA, Dawson DC, et al.
Missense mutation (G480C) in the CFTR gene associated with protein mislocalization but normal chloride channel activity.
Hum Mol Genet. 1995 Feb;4(2):269-73., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Gly480Cys The mutation, a cystelne for glycine substitution at residue 480 (G480C), was detected in a pancreatic insufficient, African-American, cystic fibrosis (CF) patient. Login to comment 4 ABCC7 p.Gly480Cys G480C was found on one additional CF chromosome and on none of 220 normal chromosomes, including 160 chromosomes from normal African-American individuals. Login to comment 5 ABCC7 p.Gly480Cys Western blot analysis and immunofluorescence studies revealed that, in 293T cells, the encoded mutant protein was not fully glycosylated and failed to reach the plasma membrane, suggesting that the G480C protein was subject to defective Intracellular processing. Login to comment 6 ABCC7 p.Gly480Cys However, in Xenopus oocytes, a system in which mutant CFTR proteins are less likely to experience an intracellular processing/trafficking deficit, expression of G480C CFTR was associated with a chloride conductance that exhibited a sensitivity to activation by forskolin and 3-isobutyl-1-methylxanthine (IBMX) that was similar to that of wild-type CFTR. Login to comment 23 ABCC7 p.Gly551Asp Some CFTR variants, such as G551D, are purely functional mutants while others, such as AF508, not only exhibit a processing defect but also possess a demonstrated functional defect when assayed under temperature conditions which allow the protein to reach the plasma membrane. Login to comment 25 ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys In this report we describe a glycine to cysteine substitution at amino acid 480 (G480C), which is associated with a mislocalized protein in mammalian cells cultured at 37°C but *To whom correspondence should be addressed at present address: Department of Physiology, University of Michigan, Ann Arbor, MI 48109-0622, USA + To whom reprint requests should be addressed at present address: National Center for Human Genome Research, National Institutes of Health, Building 38A, Room 605, Bethesda, MD 20892, USA exhibits sensitivity to activation by cAMP in Xenopus ooyctes at 19°C similar to that of wild-type CFTR. Login to comment 29 ABCC7 p.Gly480Cys The sequence revealed a G to T transversion at nucleotide 1570 (Fig. IB), creating a glycine to cysteine missense mutation at residue 480 (G480C). Login to comment 32 ABCC7 p.Gly480Cys The G480C mutation was detected in a second African-American CF patient by denaturing gradient gel electrophoresis and confirmed by sequencing. Login to comment 35 ABCC7 p.Gly480Cys G480C was detected in one additional non-AF508 CF chromosome of 378 tested. Login to comment 36 ABCC7 p.Gly480Cys This additional G480C patient has a Caucasian father and an African-American mother. Login to comment 37 ABCC7 p.Gly480Cys It could not be determined which parent carries the G480C mutation, as parental DNA was not available. Login to comment 38 ABCC7 p.Gly480Cys The three patients bearing the G480C mutation were not known to be related. Login to comment 40 ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys Immunoblot analysis and localization of G480C CFTR The effect of the G480C mutation on the processing of the CFTR protein was investigated by means of Western blot analysis and immunofluorescence. Login to comment 41 ABCC7 p.Gly480Cys 293T cells, which lack detectable endogenous CFTR (15), were transfected with wild-type, AF508 or G480C CFTR cDNA expression vectors. Login to comment 43 ABCC7 p.Gly480Cys Only the incompletely glycosylated form of CFTR was observed in cells transfected with AF5O8 or G480C cDNA. Login to comment 44 ABCC7 p.Gly480Cys Even upon longer exposures, there was no detectable signal for mature CFTR in the AF508 and G480C lysates. Login to comment 45 ABCC7 p.Gly480Cys This suggests that G480C CFTR, like AF508, is not processed to the mature, fully glycosylated form of CFTR in the cell. Login to comment 48 ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys Both AF508, a known trafficking mutant (Fig. 3B) and G480C (Fig. 3C) CFTR were associated with staining that was restricted to the cytoplasm, consistent with the classification of G480C as a trafficking mutation. Login to comment 49 ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys Functional analysis of G480C in Xenopus oocytes Membrane currents were recorded from Xenopus oocytes injected with RNA transcribed from either wild-type or G480C constructs. Login to comment 52 ABCC7 p.Gly551Asp For the G551D mutant that is processed normally in mammalian cells the severe cytstic fibrosis in patients was correlated with a substantially increased value of K\Q for activation by IBMX. Login to comment 53 ABCC7 p.Gly480Cys In contrast, in oocytes injected with RNA coding for G480C CFTR the Ki/2 for activation of Cl" conductance was identical to that for wild-type CFTR (Fig. 4). Login to comment 55 ABCC7 p.Gly480Cys DISCUSSION The identification of G480C in three CF chromosomes, including two apparently independent, unrelated African-American CF chromosomes, but not in any normal chromosomes (includ- Sllbp -2»lbp Figure 1. Login to comment 56 ABCC7 p.Gly480Cys Detection of G480C by chemical mismatch cleavage and DNA sequence analysis. Login to comment 58 ABCC7 p.Gly480Cys Lanes 1 and 2 show DNA from patients with no mutations in exon 10 and lane 3 shows DNA from a patient with the G480C mutation. Login to comment 59 ABCC7 p.Gly480Cys (B) Nucleotide sequence of a portion of exon 10 from the patient bearing the G480C mutation. Login to comment 64 ABCC7 p.Gly480Cys Lane 1, mock transfected; lane 2, wild-type transfected; lane 3, AF508 transfected; lane 4, G480C transfected; lane 5, T84. Login to comment 67 ABCC7 p.Gly480Cys ing 160 African-American normal chromosomes) is consistent with the classification of G480C as a disease causing mutation, rather than a neutral polymorphism. Login to comment 70 ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys The identification of G480C mutation in at least two African-American CF chromosomes suggests that G480C may represent a common CF mutation in the African-American population. Login to comment 73 ABCC7 p.Gly480Cys The results described here suggest that G480C CFTR, like AF508 CFTR, is misprocessed in the cell. Login to comment 74 ABCC7 p.Gly480Cys The functional properties of G480C CFTR were examined in Xenopus oocytes. Login to comment 75 ABCC7 p.Gly480Cys While expression of G480C CFTR in Xenopus oocytes is not equivalent to expression in mammalian cells at a permissive temperature, previous studies have shown that mutant CFTR proteins are less likely to be subject to intracellular trafficking problems when expressed in oocytes (11,12). Login to comment 77 ABCC7 p.Gly551Asp When expressed in Xenopus oocytes CFTRs bearing mutations like G551D or AF508 that are associated with severe disease give rise to Cl~ conductance characterized by markedly reduced sensitivity to activating conditions (11). Login to comment 78 ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys In contrast, dose-dependent activation of G480C in oocytes indicates that in this system, G480C CFTR functions identically to wild type. Login to comment 80 ABCC7 p.Gly480Cys G480C is not located in a region of the protein expected to form the conducting pore, rather it is located in the first NBF where other mutations have not proven to affect single channel conductance (13). Login to comment 81 ABCC7 p.Gly480Cys If indeed G480C is associated with functional properties that are identical to wild-type CFTR at temperatures permissive for normal trafficking, it is the first example of a CF missense Figure 3. Login to comment 82 ABCC7 p.Gly480Cys Localization of wild-type, AF508, and G480C CFTR by fluorescence miscroscopy. Login to comment 85 ABCC7 p.Gly480Cys IBMX dose-response relationships for wild-type and G480C CFTR. Login to comment 90 ABCC7 p.Gly551Asp These results demonstrate that mislocalization of CFTR is sufficient to cause disease and, when considered in the context of mutations like G551D that are functionally deficient yet processed normally, suggest that the fidelity of processing is not related to Cl~ channel function. Login to comment 109 ABCC7 p.Val1475Met ABCC7 p.Val1475Met The full length constructs contain a methionine for valine substitution at amino acid 1475 (V1475M). Login to comment 110 ABCC7 p.Val1475Met V1475M CFTR is associated with wild-type activity in a Xenopus cocyte assay (12). Login to comment