The cystic fibrosis mutation G1349D within the signature motif LSHGH of NBD2 abolishes the activation of CFTR chloride channels by genistein.
Cystic fibrosis (CF) is a common lethal genetic disease caused by autosomal recessive mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel that belongs to the ATP-Binding Cassette (ABC) family of transporters. The class III CF mutations G551D and G1349D are located within the "signature" sequence LSGGQ and LSHGH of NBD1 and NBD2, respectively. We have constructed by site-directed mutagenesis vectors encoding green fluorescent protein (GFP)-tagged wild-type (wt) CFTR or CFTR containing delF508, G551D, G1349D and G551D/G1349D to study their pharmacology after transient expression in COS-7 cells. We show that IBMX and the benzo[c]quinolizinium derivative MPB-91 stimulates the activity of G1349D-, G551D- and G551D/G1349D-CFTR only in the presence of cAMP-promoting agents like forskolin or cpt-cAMP. Similar half-maximal effective concentrations (EC(50)) of MPB-91 (22-36microM) have been determined for wt-, G551D-, G1349D- and G551D/G1349D-CFTR. The isoflavone genistein stimulates wild-type (wt)- and delF508-CFTR channel activity in a non-Michaelis-Menten manner. By contrast, the response of G1349D- and G551D-CFTR to genistein is dramatically altered. First, genistein is not able to stimulate G1349D- and G551D/G1349D-CFTR. Second, genistein stimulates G551D-CFTR without any inhibition at high concentration. We conclude from these results that whereas G551 in NBD1 is an important molecular site for inhibition of CFTR by genistein, the symmetrical G1349 in NBD2 is also one major site but for the activation of CFTR by genistein. Because both mutations alter specifically the mechanism of CFTR channel activation by genistein, we believe that the signature sequences of CFTR act as molecular switches that upon interaction with genistein turn on and off the channel.
Melin
Patricia
P
Institut de Physiologie et Biologie Cellulaires CNRS UMR 6187, Université de Poitiers, 40 Avenue du Recteur Pineau, 86022 Poitiers, France.
Thoreau
Vincent
V
Norez
Caroline
C
Bilan
Frédéric
F
Kitzis
Alain
A
Becq
Frédéric
F
D020816:Amino Acid Motifs / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D019556:COS Cells; D003550:Cystic Fibrosis / Q000473:pathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D019833:Genistein / Q000494:pharmacology*; D005998:Glycine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D014162:Transfection
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D001224:Aspartic Acid; D019833:Genistein; D005998:Glycine