Molecular assembly of cystic fibrosis transmembrane conductance regulator in plasma membrane.
Based on electrophysiological measurements, it has been argued that the active form of cystic fibrosis trans-membrane conductance regulator (CFTR) Cl(-) channel is a multimer. It has also been demonstrated that this multimerization is likely due to PDZ domain-interacting partners. Here we demonstrate that although CFTR in vitro can self-associate into multimers, which depends on PDZ-based interactions, this may not be the case in cell membrane. Using chemical cross-linking, we demonstrated that CFTR exists as a higher order complex in cell membrane. However, this higher order complex is predominantly CFTR dimers, and the PDZ-interacting partners (Na(+)/H(+) exchanger regulatory factor-1 (NHERF1) and NHERF2) constitute approximately 2% of this complex. Interestingly solubilizing membrane expressing CFTR in detergents such as Triton X-100, Nonidet P-40, deoxycholate, and SDS tended to destabilize the CFTR dimers and dissociate them into monomeric form. The dimerization of CFTR was tightly regulated by cAMP-dependent protein kinase-dependent phosphorylation and did not depend on the active form of the channel. In addition, the dimerization was not influenced by either the PDZ motif or its interacting partners (NHERF1 and NHERF2). We also demonstrated that other signaling-related proteins such as Gbeta and syntaxin 1A can be in this higher order complex of CFTR as well. Our studies provide a deeper understanding of how the CFTR assembly takes place in native cell membrane.
Li
Chunying
C
Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.
Roy
Koushik
K
Dandridge
Keanna
K
Naren
Anjaparavanda P
AP
D020816:Amino Acid Motifs; D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry; D019921:Biotinylation; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis / Q000737:chemistry*; D003598:Cytoskeletal Proteins / Q000737:chemistry; D003902:Detergents / Q000494:pharmacology; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004594:Electrophysiology; D006801:Humans; D009419:Nerve Tissue Proteins / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D017923:Sodium-Hydrogen Exchangers; D013347:Subcellular Fractions / Q000378:metabolism; D050827:Syntaxin 1
D000954:Antigens, Surface; C505032:CFTR protein, human; D003432:Cross-Linking Reagents; D003598:Cytoskeletal Proteins; D003902:Detergents; D009419:Nerve Tissue Proteins; D010455:Peptides; D010750:Phosphoproteins; C494212:STX1A protein, human; D017923:Sodium-Hydrogen Exchangers; D050827:Syntaxin 1; C093376:sodium-hydrogen exchanger regulatory factor; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP