ABCMdb

PMID: 10652362

Weixel KM, Bradbury NA
The carboxyl terminus of the cystic fibrosis transmembrane conductance regulator binds to AP-2 clathrin adaptors.
J Biol Chem. 2000 Feb 4;275(5):3655-60., 2000-02-04 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Tyr1424Ala Substitution of an alanine residue for tyrosine at position 1424 significantly reduced the ability of AP-2 to bind the carboxyl terminus of CFTR; however, mutation to a phenylalanine residue (an amino acid found at position 1424 in dogfish CFTR) did not perturb AP-2 binding. Login to comment 93 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala To examine the contribution of Tyr1424 to the association between the carboxyl terminus of CFTR and AP-2 adaptors, Tyr1424 was mutated to Ala in the context of the GST fusion protein GST-CT (GST-CT-Y1424A). Login to comment 94 ABCC7 p.Tyr1424Ala GST-CT-Y1424A preabsorbed to glutathione-Sepharose was incubated with purified adaptors, and after extensive washing, bound proteins were analyzed by immunoblot as before. Login to comment 95 ABCC7 p.Tyr1424Ala Although GST-CT-Y1424A was capable of binding AP-2 complexes (Fig. 6A), its capacity for binding was significantly reduced compared with wild-type GST-CT. Login to comment 96 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala Densitometric analysis of the immunoblot indicates that the Y1424A mutation reduces binding to AP-2 complexes compared with the wild-type carboxyl terminus construct, GST-CT, even at higher amounts of GST-CT-Y1424A (Fig. 6B). Login to comment 99 ABCC7 p.Tyr1424Phe GST-CT-Y1424F was able to bind AP-2 complexes with as much efficiency as wild-type GST-CT, suggesting that the phenylalanine at position 1424 is capable of participating in the endocytosis signal (Fig. 6B). Login to comment 100 ABCC7 p.Tyr1424Ala When the Student`s t test was applied to the data in Fig. 6, GST-CF- Y1424A showed significant reduction of adaptor binding compared with wild-type constructs, with a p value of Ͻ0.02. Login to comment 101 ABCC7 p.Tyr1424Phe In contrast, no significant difference was observed between GST-CT-Y1424F and wild-type constructs. A Peptide Derived from the Cytoplasmic Tail of CFTR Inhibits CFTR-AP-2 Binding-To investigate further the role of the carboxyl terminus of CFTR as a docking site for AP-2 adaptors, a peptide representing a cytoplasmic domain sequence of CFTR was tested for its ability to block AP-2 binding to GST-CT constructs. A 19-residue peptide, Y19P (CQQFLVIEEN- KVRQYDSIQ), was synthesized corresponding to amino acids 1410-1428 of CFTR. Login to comment 109 ABCC7 p.Tyr1424Ala Mutation of tyrosine 1424 to an alanine causes marked loss of beta-structure at this site (Fig. 8). Login to comment 148 ABCC7 p.Tyr1424Ala Mutation of tyrosine 1424 to alanine but not phenylalanine inhibits AP-2 binding to CFTR. Login to comment 150 ABCC7 p.Tyr1424Ala 10 or 20 ␮g of wild-type (WT, lanes 1 and 2) or Y1424A (lanes 3 and 4) constructs were incubated with purified adaptor complexes. Login to comment 152 ABCC7 p.Tyr1424Ala The asterisk indicates p Ͻ 0.02 for difference between Y1424A and wild type by Student`s t test. Login to comment 154 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Phe 20 ␮g of Y1424F protein (lane 1), Y1424A (lane 2), wild-type (lane 3), or GST alone (lane 4) were incubated with purified adaptors as described. Login to comment 162 ABCC7 p.Tyr1424Ala Mutation of Tyr1424 to Ala resulted in a significant inhibition of AP-2 binding to the CFTR-GST fusion protein, indicating the importance of this residue in AP-2 binding. Login to comment 163 ABCC7 p.Tyr1424Ala However, binding of AP-2 to Y1424A-CFTR was not completely abolished. Login to comment 166 ABCC7 p.Tyr1424Ala ABCC7 p.Tyr1424Ala Of note are the observations of Collawn and colleagues (25), who have shown that a Y1424A mutation in transiently expressed CFTR results in a 40% inhibition of CFTR endocytosis rates, a finding consistent with the extent to which Y1424A mutations inhibit AP-2 binding. Login to comment 169 ABCC7 p.Tyr1424Phe Such is also the case for CFTR, as a Y1424F mutation resulted in a construct that was unimpaired in its ability to bind AP-2 adaptors. Login to comment