Increased diffusional mobility of CFTR at the plasma membrane after deletion of its C-terminal PDZ binding motif.
The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated Cl- channel expressed at the apical plasma membrane. It has been proposed that the C-terminal PDZ binding motif of CFTR is required for its apical membrane targeting and that PDZ-domain interactions may tether CFTR to the actin cytoskeleton via soluble proteins including EBP50/NHERF1 and ezrin. We measured the diffusional mobility of human CFTR in the plasma membrane of Madin-Darby canine kidney cells by photobleaching of green fluorescent protein (GFP)-CFTR chimeras. After bleaching by a focused laser beam, GFP-CFTR fluorescence in the bleached membrane region recovered to approximately 90% of its initial level, indicating that nearly all of the CFTR was mobile. The GFP-CFTR diffusion coefficient (D) was 0.99 +/- 0.09 x 10(-10) cm2/s at 37 degrees C, similar to that of other membrane proteins. GFP-CFTR diffusion was not altered by protein kinase A or C activators but was blocked by paraformaldehyde and filipin. CFTR mutants lacking functional PDZ-binding domains (GFPCFTR-DeltaTRL and GFP-CFTR-DeltaTRA) were also mobile with D significantly increased by approximately 60% compared with GFP-CFTR. However, GFP-CFTR, GFP-CFTR-Delta TRL, and GFP-CFTR-DeltaTRA had similar mobilities (D approximately 12 x 10(-10) cm2/s) at the endoplasmic reticulum in brefeldin A-treated cells. Agents that modulate the actin cytoskeleton (cytochalasin D and jasplakinolide) altered the plasma membrane mobility of CFTR but not CFTR- DeltaTRL. EBP50 (NHERF1), a PDZ domain-containing protein that interacts with the C terminus of CFTR, diffused freely in the cytoplasm with a diffusion coefficient of 0.9 +/- 0.1 x 10(-7) cm2/s. EBP50 diffusion increased by approximately 2-fold after deletion of its ezrin-binding domain. These results indicate that wild-type CFTR is not tethered statically at the plasma membrane but that its diffusion is dependent on PDZ-domain interactions and an intact actin skeleton. PDZ-domain interactions of CFTR are thus dynamic and occur on a time scale of seconds or faster.
Haggie
Peter M
PM
Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521, USA.
Stanton
Bruce A
BA
Verkman
A S
AS
D000199:Actins / Q000737:chemistry / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004058:Diffusion; D004285:Dogs; D004789:Enzyme Activation; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013997:Time Factors; D014162:Transfection
D000199:Actins; C505032:CFTR protein, human; D018076:DNA, Complementary; D008164:Luminescent Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C