Activation mechanisms for the cystic fibrosis transmembrane conductance regulator protein involve direct binding of cAMP |
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Authors: | Pereira Malcolm M C Parker Jody Stratford Fiona L L McPherson Margaret Dormer Robert L |
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Institution: | Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK |
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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. |
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