Activation mechanisms for the cystic fibrosis transmembrane conductance regulator protein involve direct binding of cAMP

Malcolm Mc Pereira , Jody Parker , Fiona Ll Stratford , Margaret Mcpherson , Robert L Dormer (1)

1 Medical Biochemistry & Immunology

The cystic fibrosis transmembrane conductance regulator (CFTR) 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 nucleotide binding- and R- domains of CFTR (NBD1-R) was synthesised to determine directly whether CFTR binds cyclic AMP. 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 [ 3}H]-ATP by non-radioactive ATP at 3.5µM and 3.1mM was demonstrated. [ 3}H]-cyclic AMP bound to the protein with different affinities than for ATP (half-maximal displacement by cyclic AMP at 2.6µM and 167µM). Introduction of a mutation (T421A) in a motif predicted to be important for cyclic nucleotide binding decreased the higher affinity binding of cyclic AMP to 9.2µM. The anti-CFTR antibody (MPNB) that inhibits CFTR-mediated protein secretion also inhibited cyclic AMP binding. Thus, binding of cyclic AMP to CFTR is consistent with a role in activation of protein secretion, a process defective in cystic fibrosis gland cells. Furthermore, the binding site may be important in the mechanism by which drugs activate mutant CFTR and correct defective ΔF508-CFTR trafficking.

Mots clés: Life Sciences

Malcolm Mc Pereira, Jody Parker, Fiona Ll Stratford, Margaret Mcpherson, Robert L Dormer. Activation mechanisms for the cystic fibrosis transmembrane conductance regulator protein involve direct binding of cAMP. Biochemical Journal, 2007, 405 (1), pp.181-189. ⟨10.1042/BJ20061879⟩. ⟨hal-00478722⟩