title: Activation mechanisms for the cystic fibrosis transmembrane conductance regulator protein involve direct binding of cAMP.
abstract: The CFTR [CF (cystic fibrosis) transmembrane conductance regulator] chloride channel is activated by cyclic nucleotide-dependent phosphorylation and ATP binding, but also by non-phosphorylation-dependent mechanisms. Other CFTR functions such as regulation of exocytotic protein secretion are also activated by cyclic nucleotide elevating agents. A soluble protein comprising the first NBD (nucleotide-binding domain) and R-domain of CFTR (NBD1-R) was synthesized to determine directly whether CFTR binds cAMP. An equilibrium radioligand-binding assay was developed, firstly to show that, as for full-length CFTR, the NBD1-R protein bound ATP. Half-maximal displacement of [3H]ATP by non-radioactive ATP at 3.5 microM and 3.1 mM was demonstrated. [3H]cAMP bound to the protein with different affinities from ATP (half-maximal displacement by cAMP at 2.6 and 167 microM). Introduction of a mutation (T421A) in a motif predicted to be important for cyclic nucleotide binding decreased the higher affinity binding of cAMP to 9.2 microM. The anti-CFTR antibody (MPNB) that inhibits CFTR-mediated protein secretion also inhibited cAMP binding. Thus binding of cAMP to CFTR is consistent with a role in activation of protein secretion, a process defective in CF gland cells. Furthermore, the binding site may be important in the mechanism by which drugs activate mutant CFTR and correct defective DeltaF508-CFTR trafficking.
authors: Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK
authors: Malcolm M C
authors: MM
authors: Pereira
authors: Jody
authors: J
authors: Parker
authors: Fiona L L
authors: FL
authors: Stratford
authors: Margaret
authors: M
authors: McPherson
authors: Robert L
authors: RL
authors: Dormer
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism*; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000906:Antibodies; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D006152:Cyclic GMP