ABCMdb

PMID: 10852925

Moyer BD, Duhaime M, Shaw C, Denton J, Reynolds D, Karlson KH, Pfeiffer J, Wang S, Mickle JE, Milewski M, Cutting GR, Guggino WB, Li M, Stanton BA
The PDZ-interacting domain of cystic fibrosis transmembrane conductance regulator is required for functional expression in the apical plasma membrane.
J Biol Chem. 2000 Sep 1;275(35):27069-74., 2000-09-01 [PubMed]

Sentences

No. Mutations Sentence Comment

36 ABCC7 p.Ser1455* EXPERIMENTAL PROCEDURES Expression Vectors-pGFP-CFTR, pGFP-CFTR-⌬TRL, and pGFP-CFTR-S1455X, encoding enhanced GFP fused to the N terminus of wt-CFTR, CFTR lacking the 3 C-terminal amino acids, and CFTR lacking the 26 C-terminal amino acids, respectively, were constructed as described previously (23, 29). Login to comment 81 ABCC7 p.Ser1455* By contrast, EBP50 could not be co-immunoprecipitated with CFTR-QDTRA or CFTR-⌬TRL, which are nonpolarized, and, as shown previously, CFTR-S1455X (23) (Fig. 2 and Table I). Login to comment 82 ABCC7 p.Ser1455* CFTR-S1455X, a naturally occurring mutation that lacks the 26 C-terminal amino acids, is polarized to the lateral membrane (23). Login to comment 92 ABCC7 p.Ser1455* By contrast, in cells stably expressing mutant CFTRs that were not polarized to the apical plasma membrane, including CFTR-⌬TRL, CFTR-S1455X and CFTR-QDTRA, FIG. 1. Login to comment 104 ABCC7 p.Ser1455* CFTR AP/BL ratio Co-IP EBP50 Isc % ␮A/cm2 wt-CFTR 10.4 Ϯ 2.0 100 8.3 Ϯ 0.8 ⌬TRL 0.6 Ϯ 0.3a 0a 2.6 Ϯ 0.4a QDTRA 0.7 Ϯ 0.2a 0a 2.0 Ϯ 0.3a QDTAL 10.8 Ϯ 1.8 40a - QDARL 10.9 Ϯ 2.0 10a 3.6 Ϯ 0.6a QDVRL 11.2 Ϯ 1.9 16a 4.8 Ϯ 0.5a QATRL 15.2 Ϯ 2.0 74 8.6 Ϯ 0.9 ADTRL 11.4 Ϯ 2.0 78 7.6 Ϯ 1.5 S1455X 0.2 Ϯ 0.1a 0a 1.7 Ϯ 0.2a a Indicates cAMP-stimulated chloride secretion was not significantly different from parental, untransfected cells (Fig. 3). Login to comment 114 ABCC7 p.Ser1455* Similar inefficient expression was observed for CFTR-S1455X, CFTR-QDTRA, CFTR-QDVRL and CFTR-QDARL (2-5% of total CFTR was expressed in the plasma membranes). Login to comment 129 ABCC7 p.Ser1455* By contrast, CFTR proteins that did not associate with EBP50, including QDTRA (* indicates protein could not be detected) and, as shown previously, S1455X and ⌬TRL (23), were not polarized to the apical membrane (Fig. 1). Login to comment 141 ABCC7 p.Ser1455* By contrast, CFTR mutants that were not polarized to the apical membrane, including ⌬TRL, S1455X, and QDTRA, did not produce cAMP-stimulated chloride currents significantly different from cAMP-stimulated currents in untransfected, parental cells. Login to comment 144 ABCC7 p.Ser1455* Number of monolayers studied was: 14 for parental cells, 22 for wt-CFTR, 14 for ⌬TRL, 8 for S1455X, 21 for QDTRA, 14 for QDVRL, 17 for QDARL, 5 for QATRL, and 4 for ADTRL. Login to comment