Modulation of mature cystic fibrosis transmembrane regulator protein by the PDZ domain protein CAL.
We have previously identified the cystic fibrosis transmembrane regulator (CFTR)-interacting protein CAL and demonstrated that CAL modulates CFTR plasma membrane expression by retaining CFTR within the cell. Here, we report that in addition to regulating membrane expression, CAL also regulates the expression of mature CFTR. The co-expression of hemagglutinin-tagged or Myc-tagged CAL with green fluorescent protein (GFP)-CFTR in COS-7 cells causes a dose-dependent reduction in mature GFP-CFTR, independent of its tags. Bafilomycin A1, a lysosomal proton pump inhibitor, increases mature GFP-CFTR, confirming previous reports of lysosomal degradation of mature CFTR. Importantly, bafilomycin A1 reverses CAL-mediated CFTR degradation. The proteasome inhibitor, MG132, on the other hand, does not reverse the effect of CAL. CAL has no effect on CFTR maturation, suggesting that it exerts its effects on mature CFTR. Co-expression of CAL enhances the degradation of CFTR. We showed previously that CAL reduces the half-life of CFTR at the cell surface. Here we show that expression of dominant-negative dynamin 2 K44A, a large GTPase inhibitor that is known to inhibit clathrin-mediated endocytosis and vesicle formation in the Golgi, increases cell surface CFTR as measured by surface biotinylation. More importantly, dynamin 2 K44A also restores cell surface CFTR in CAL-overexpressing cells and partially blocks the CAL-mediated degradation of mature CFTR. These data suggest a model in which CAL retains CFTR in the cell and targets CFTR for degradation.
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Jie
J
Cheng
Hua
H
Wang
William B
WB
Guggino
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000032:analysis / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000032:analysis / Q000378:metabolism*; D034285:Dynamin II / Q000502:physiology; D000077614:Golgi Matrix Proteins; D006801:Humans; D018942:Macrolides / Q000494:pharmacology; D008565:Membrane Proteins / Q000032:analysis / Q000502:physiology*; D026901:Membrane Transport Proteins; D021381:Protein Transport; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; C505032:CFTR protein, human; D002352:Carrier Proteins; C434484:GOPC protein, human; D000077614:Golgi Matrix Proteins; D018942:Macrolides; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C040929:bafilomycin A1; D034285:Dynamin II