ABCMdb

PMID: 22399289

Cihil KM, Ellinger P, Fellows A, Stolz DB, Madden DR, Swiatecka-Urban A
Disabled-2 protein facilitates assembly polypeptide-2-independent recruitment of cystic fibrosis transmembrane conductance regulator to endocytic vesicles in polarized human airway epithelial cells.
J Biol Chem. 2012 Apr 27;287(18):15087-99. Epub 2012 Mar 7., [PubMed]

Sentences

No. Mutations Sentence Comment

88 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala Plasmids and Transient Transfection-The cDNAs encoding human full-length WT-CFTR and the CFTR Y1424A mutant in pS65T-C1 vector (GFP-CFTR WT and GFP-CFTR Y1424A, respectively) were generous gifts from Dr. Bruce A. Stanton, Department of Physiology, Dartmouth Medical School, Hanover, NH. Human Dab2 (Uniprot accession number P98082-1) was obtained from Origene (SC321375) Technologies, Inc., Rockville, MD). Login to comment 89 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala Plasmids and Transient Transfection-The cDNAs encoding human full-length WT-CFTR and the CFTR Y1424A mutant in pS65T-C1 vector (GFP-CFTR WT and GFP-CFTR Y1424A, respectively) were generous gifts from Dr. Bruce A. Stanton, Department of Physiology, Dartmouth Medical School, Hanover, NH. Human Dab2 (Uniprot accession number P98082-1) was obtained from Origene (SC321375) Technologies, Inc., Rockville, MD). Login to comment 253 ABCC7 p.Tyr1424Ala First, we examined whether the Y1424A substitution in CFTR, previously shown to disrupt the CFTR-òe;2 interaction, affects CFTR recruitment to CCVs. Login to comment 254 ABCC7 p.Tyr1424Ala Parental CFBE41o-cells transiently transfected with the GFP-CFTR WT or the GFP-CFTR Y1424A mutant were grown in plastic tissue culture dishes for 72 h. Login to comment 255 ABCC7 p.Tyr1424Ala First, we examined whether the Y1424A substitution in CFTR, previously shown to disrupt the CFTR-␮2 interaction, affects CFTR recruitment to CCVs. Login to comment 256 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala Parental CFBE41o-cells transiently transfected with the GFP-CFTR WT or the GFP-CFTR Y1424A mutant were grown in plastic tissue culture dishes for 72 h. Login to comment 257 ABCC7 p.Tyr1424Ala Next, we examined the effect of the Y1424A substitution on CFTR endocytosis. Login to comment 258 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala As illustrated in Fig. 8, the abundance of the GFP-CFTR WT and GFP-CFTR Y1424A in CCVs was similar. Login to comment 259 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala Next, we examined the effect of the Y1424A substitution on CFTR endocytosis. Login to comment 260 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala The biotinylation-based endocytic assays were conducted in parental CFBE41o-cells expressing GFP-CFTR WT or the GFP-CFTR Y1424A mutant. Login to comment 261 ABCC7 p.Tyr1424Ala As illustrated in Fig. 9, endocytosis of the GFP-CFTR WT and GFP-CFTR Y1424A was similar in the linear uptake phase, consistent with steady-state measurements (Figs. 8 and 10). Login to comment 262 ABCC7 p.Tyr1424Ala Finally, studies were conducted to determine the effect of the Y1424A substitution on CFTR stability in the plasma membrane. Login to comment 273 ABCC7 p.Tyr1424Ala Error bars, S.E. Dab2 Facilitates CFTR Endocytosis APRIL 27, 2012ߦVOLUME 287ߦNUMBER 18 JOURNAL OF BIOLOGICAL CHEMISTRY 15093 at SEMMELWEIS UNIV OF MEDICINE on December 5, membrane biotinylation at &#b0;C, the disappearance of the GFP-CFTR WT or GFP-CFTR Y1424A mutant from the plasma membrane was monitored as a function of time at 37 &#b0;C. Login to comment 274 ABCC7 p.Tyr1424Ala As shown in Fig. 10, the plasma membrane stability of the GFP-CFTR WT and GFP-CFTR Y1424A was similar. Login to comment 275 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala Error bars, S.E. Dab2 Facilitates CFTR Endocytosis APRIL 27, 2012•VOLUME 287•NUMBER 18 JOURNAL OF BIOLOGICAL CHEMISTRY 15093 membrane biotinylation at °C, the disappearance of the GFP-CFTR WT or GFP-CFTR Y1424A mutant from the plasma membrane was monitored as a function of time at 37 °C. Login to comment 276 ABCC7 p.Tyr1424Ala As shown in Fig. 10, the plasma membrane stability of the GFP-CFTR WT and GFP-CFTR Y1424A was similar. Login to comment 277 ABCC7 p.Tyr1424Ala The above data demonstrate that the Y1424A mutation affects neither CFTR recruitment to CCVs, CFTR endocytosis, nor CFTR stability in the plasma membrane and suggest that the CFTR interaction with ␮2 adaptin is not essential for CFTR endocytosis in human airway epithelial cells. Login to comment 325 ABCC7 p.Tyr1424Ala Subcellular fractionation experiments performed to determine the effect of the Y1424A mutation on CFTR recruitment CCV in CFBE41o-cells. Login to comment 326 ABCC7 p.Tyr1424Ala The CCV fractions were prepared by density gradient and differential centrifugation in parental CFBE41o-cells 72 h after transfection with the GFP-CFTR WT or GFP-CFTR Y1424A mutant. Login to comment 327 ABCC7 p.Tyr1424Ala Subcellular fractionation experiments performed to determine the effect of the Y1424A mutation on CFTR recruitment CCV in CFBE41o-cells. Login to comment 328 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala The CCV fractions were prepared by density gradient and differential centrifugation in parental CFBE41o-cells 72 h after transfection with the GFP-CFTR WT or GFP-CFTR Y1424A mutant. Login to comment 330 ABCC7 p.Tyr1424Ala A representative Western blot (B) and summary of experiments (C) demonstrate that the Y1424A mutation did not decrease the abundance of CFTR in CCVs. Login to comment 354 ABCC7 p.Tyr1424Ala Endocytic assays performed to determine the effect of the Y1424A mutation on CFTR endocytosis. Login to comment 355 ABCC7 p.Tyr1424Ala Endocytic assays were performed in parental CFBE41o-cells 72 h after transfection with the GFP-CFTR WT or GFP-CFTR Y1424A mutant. Login to comment 356 ABCC7 p.Tyr1424Ala Endocytic assays performed to determine the effect of the Y1424A mutation on CFTR endocytosis. Login to comment 357 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala Endocytic assays were performed in parental CFBE41o-cells 72 h after transfection with the GFP-CFTR WT or GFP-CFTR Y1424A mutant. Login to comment 359 ABCC7 p.Tyr1424Ala A representative Western blot (IB; A) and summary of experiments (B) demonstrates that the Y1424A mutation did not inhibit CFTR endocytosis. Login to comment 364 ABCC7 p.Tyr1424Ala Biotinylation experiments performed to determine the effects of the Y1424A mutation on CFTR expression in the plasma membrane of CFBE41o-cells as a function of time. Login to comment 365 ABCC7 p.Tyr1424Ala Biotinylation experiments were performed in parental CFBE41o-cells 72 h after transfection with the GFP-CFTR WT or GFP-CFTR Y1424A mutant. Login to comment 366 ABCC7 p.Tyr1424Ala Biotinylation experiments performed to determine the effects of the Y1424A mutation on CFTR expression in the plasma membrane of CFBE41o-cells as a function of time. Login to comment 367 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala Biotinylation experiments were performed in parental CFBE41o-cells 72 h after transfection with the GFP-CFTR WT or GFP-CFTR Y1424A mutant. Login to comment 369 ABCC7 p.Tyr1424Ala A representative Western blot (IB; A) and summary of experiments (B) demonstrate that the Y1424A mutation did not increase CFTR stability in the plasma membrane. Login to comment