ABCMdb

PMID: 16196493

Loo TW, Bartlett MC, Clarke DM
Rescue of DeltaF508 and other misprocessed CFTR mutants by a novel quinazoline compound.
Mol Pharm. 2005 Sep-Oct;2(5):407-13., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Arg258Gly ABCC7 p.Ser945Leu ABCC7 p.His949Tyr Incubation of BHK cells stably expressing human ∆F508 CFTR with 1-10 µM CFcor-325 resulted in maturation and delivery of a functional molecule to the cell surface as determined by the iodide efflux assay. The misprocessed CFTR mutants R258G, S945L, and H949Y were also rescued by CFcor-325 in either BHK or HEK 293 cells. Login to comment 24 ABCC7 p.Gly149Arg ABCC7 p.Arg258Gly ABCC7 p.Ser945Leu ABCC7 p.His949Tyr ABCC7 p.His139Arg Wild-type, ∆F508, H139R, G149R, R258G, S945L, and H949Y CFTR cDNAs were inserted into the pcDNA3 (Invitrogen, Oakville, ON) vector as described previously.13,14 Wild-type and mutant G268V P-gp cDNAs were inserted into the pMT21 vector (Genetics Institute) as described previously.15 Baby hamster kidney (BHK) cells stably expressing CFTR or P-gp were generated by cotransfection with cDNA and pWL-neo (Stratagene, Cedar Creek, TX). Login to comment 26 ABCC7 p.Gly149Arg ABCC7 p.Arg258Gly ABCC7 p.Ser945Leu ABCC7 p.His949Tyr ABCC7 p.His139Arg Wild-type, ∆F508, H139R, G149R, R258G, S945L, and H949Y CFTR cDNAs were inserted into the pcDNA3 (Invitrogen, Oakville, ON) vector as described previously.13,14 Wild-type and mutant G268V P-gp cDNAs were inserted into the pMT21 vector (Genetics Institute) as described previously.15 Baby hamster kidney (BHK) cells stably expressing CFTR or P-gp were generated by cotransfection with cDNA and pWL-neo (Stratagene, Cedar Creek, TX). Login to comment 130 ABCC7 p.Gly149Arg ABCC7 p.Arg258Gly ABCC7 p.Ser945Leu ABCC7 p.His949Tyr ABCC7 p.His139Arg (C) BHK cells expressing misprocessed CFTR mutants H139R, G149R, R258G, S945L, H949Y, or wild-type CFTR were incubated for 48 h with (+) or without (-) 3 µM CFcor-325. Whole cell extracts were subjected to immunoblot analysis with a rabbit polyclonal antibody against CFTR. Login to comment 132 ABCC7 p.Gly149Arg ABCC7 p.Arg258Gly ABCC7 p.Ser945Leu ABCC7 p.His949Tyr ABCC7 p.His139Arg (C) BHK cells expressing misprocessed CFTR mutants H139R, G149R, R258G, S945L, H949Y, or wild-type CFTR were incubated for 48 h with (+) or without (-) 3 µM CFcor-325. Whole cell extracts were subjected to immunoblot analysis with a rabbit polyclonal antibody against CFTR. Login to comment 142 ABCC7 p.Gly149Arg ABCC7 p.Arg258Gly ABCC7 p.Ser945Leu ABCC7 p.His949Tyr ABCC7 p.His139Arg BHK cells expressing mutants H139R, G149R, and R258G in the first transmembrane domain (TMD1)30 or mutants S945L and H949Y in TMD213 were treated with or without 3 µM CFcor-325 for 48 h. Whole cell SDS extracts were then subjected to immunoblot analysis. Login to comment 143 ABCC7 p.Arg258Gly ABCC7 p.Ser945Leu ABCC7 p.His949Tyr The presence of CFcor-325 significantly enhanced maturation of mutants R258G, S945L, and H949Y (Figure 2C). Login to comment 144 ABCC7 p.Gly149Arg ABCC7 p.Gly149Arg ABCC7 p.Arg258Gly ABCC7 p.Ser945Leu ABCC7 p.His949Tyr ABCC7 p.His139Arg ABCC7 p.His139Arg BHK cells expressing mutants H139R, G149R, and R258G in the first transmembrane domain (TMD1)30 or mutants S945L and H949Y in TMD213 were treated with or without 3 µM CFcor-325 for 48 h. Whole cell SDS extracts were then subjected to immunoblot analysis. Login to comment 145 ABCC7 p.Arg258Gly ABCC7 p.Ser945Leu ABCC7 p.His949Tyr The presence of CFcor-325 significantly enhanced maturation of mutants R258G, S945L, and H949Y (Figure 2C). Login to comment 146 ABCC7 p.Gly149Arg ABCC7 p.His139Arg By contrast, CFcor-325 had little effect on mutants H139R or G149R. Login to comment 147 ABCB1 p.Gly268Val ABCB1 p.Gly268Val ABCB1 p.Gly268Val An explanation for the ability of CFcor-325 to promote folding and trafficking of misprocessed CFTR mutants is that it diffused into the ER and directly interacts with the mutant protein during biogenesis as observed with P-gp.31,32 Another explanation is that CFcor-325 has a nonspecific effect on protein folding similar to that observed when low temperature or osmolytes such as glycerol or trimethylamine N-oxide are used to rescue misfolded proteins.9,10 To test whether CFcor-325 had a direct or indirect effect on protein folding, we used the P-gp processing mutant G268V as a control.33 We used the G268V P-gp mutant, rather than mutant ∆Y490 P-gp (equivalent to ∆F508 in CFTR), as a control because maturation of the G268V mutant is not promoted by growth at low temperature (27 °C) or by osmolytes such as glycerol and trimethylamine N-oxide (unpublished observations). Login to comment 148 ABCB1 p.Gly268Val ABCB1 p.Gly268Val Maturation of mutant G268V to yield active enzyme at the cell surface, however, can be achieved by carrying out expression in the presence of drug substrates or modulators.34 Accordingly, BHK cells stably expressing P-gp mutant G268V were incubated with 0-10 µM CFcor-325 for 48 h. Login to comment 149 ABCB1 p.Gly268Val ABCB1 p.Gly268Val ABCB1 p.Gly268Val An explanation for the ability of CFcor-325 to promote folding and trafficking of misprocessed CFTR mutants is that it diffused into the ER and directly interacts with the mutant protein during biogenesis as observed with P-gp.31,32 Another explanation is that CFcor-325 has a nonspecific effect on protein folding similar to that observed when low temperature or osmolytes such as glycerol or trimethylamine N-oxide are used to rescue misfolded proteins.9,10 To test whether CFcor-325 had a direct or indirect effect on protein folding, we used the P-gp processing mutant G268V as a control.33 We used the G268V P-gp mutant, rather than mutant ∆Y490 P-gp (equivalent to ∆F508 in CFTR), as a control because maturation of the G268V mutant is not promoted by growth at low temperature (27 °C) or by osmolytes such as glycerol and trimethylamine N-oxide (unpublished observations). Login to comment 150 ABCB1 p.Gly268Val ABCB1 p.Gly268Val Maturation of mutant G268V to yield active enzyme at the cell surface, however, can be achieved by carrying out expression in the presence of drug substrates or modulators.34 Accordingly, BHK cells stably expressing P-gp mutant G268V were incubated with 0-10 µM CFcor-325 for 48 h. Login to comment 162 ABCB1 p.Gly268Val Effect of CFcor-325 on expression of misprocessed P-gp mutant G268V and on P-gp-mediated drug resistance. Login to comment 163 ABCB1 p.Gly268Val (A) BHK cells stably expressing P-gp mutant G268V were treated for 48 h with 0-10 µM CFcor-325. Whole cell extracts were then subjected to immunoblot analysis with rabbit polyclonal antibody against P-gp. Login to comment 164 ABCB1 p.Gly268Val Effect of CFcor-325 on expression of misprocessed P-gp mutant G268V and on P-gp-mediated drug resistance. Login to comment 165 ABCB1 p.Gly268Val (A) BHK cells stably expressing P-gp mutant G268V were treated for 48 h with 0-10 µM CFcor-325. Whole cell extracts were then subjected to immunoblot analysis with rabbit polyclonal antibody against P-gp. Login to comment 168 ABCB1 p.Gly268Val The ability of CFcor-325 to rescue P-gp mutant G268V suggested that it was a substrate or modulator of P-gp. Login to comment 170 ABCB1 p.Gly268Val The ability of CFcor-325 to rescue P-gp mutant G268V suggested that it was a substrate or modulator of P-gp. Login to comment 176 ABCC7 p.Arg258Gly ABCC7 p.Ser945Leu ABCC7 p.His949Tyr It was possible to promote maturation of some mutants that had mutations in different domains of CFTR including NBD1 (∆F508), TMD1 (R258G in the second intracellular loop), and TMD2 (S945L and H949Y in the third intracellular loop). Login to comment 178 ABCC7 p.Arg258Gly ABCC7 p.Ser945Leu ABCC7 p.His949Tyr It was possible to promote maturation of some mutants that had mutations in different domains of CFTR including NBD1 (∆F508), TMD1 (R258G in the second intracellular loop), and TMD2 (S945L and H949Y in the third intracellular loop). Login to comment 181 ABCC7 p.Gly149Arg ABCC7 p.His139Arg These include the substituted benzo[c]- quinolizinium compounds,39 thapsigargin,40 and curcumin.41 Drug rescue of ∆F508 CFTR with benzo[c]quinolizinium compounds, however, requires concentrations that are about 100-fold higher (250-500 µM) than that needed for CFcor-325, while rescue with thapsigargin and curcumin could not be repeated by other investigators.25,42,43 A possible explanation for the observation that curcumin treatment increased cAMP-stimulated iodide efflux in BHK cells expressing ∆F508 CFTR41 was that the compound can directly stimulate CFTR channels that were already present at the cell surface.44 We were unable to induce maturation of misprocessed CFTR mutants that had the mutations H139R or G149R in the first intracellular loop. Login to comment 183 ABCC7 p.Gly149Arg ABCC7 p.His139Arg These include the substituted benzo[c]- quinolizinium compounds,39 thapsigargin,40 and curcumin.41 Drug rescue of ∆F508 CFTR with benzo[c]quinolizinium compounds, however, requires concentrations that are about 100-fold higher (250-500 µM) than that needed for CFcor-325, while rescue with thapsigargin and curcumin could not be repeated by other investigators.25,42,43 A possible explanation for the observation that curcumin treatment increased cAMP-stimulated iodide efflux in BHK cells expressing ∆F508 CFTR41 was that the compound can directly stimulate CFTR channels that were already present at the cell surface.44 We were unable to induce maturation of misprocessed CFTR mutants that had the mutations H139R or G149R in the first intracellular loop. Login to comment 205 ABCC7 p.Gly149Arg ABCC7 p.His139Arg (44) Berger, A. L.; Randak, C. O.; Ostedgaard, L. S.; Karp, P. H.; Vermeer, D. W.; Welsh, M. J. Curcumin stimulates cystic fibrosis transmembrane conductance regulator Cl- channel activity. J. Biol. Chem. 2005, 280, 5221-5226. in the first cytoplasmic loop (H139R or G149R) prevents the establishment of these interactions. Login to comment 207 ABCC7 p.Gly149Arg ABCC7 p.His139Arg (44) Berger, A. L.; Randak, C. O.; Ostedgaard, L. S.; Karp, P. H.; Vermeer, D. W.; Welsh, M. J. Curcumin stimulates cystic fibrosis transmembrane conductance regulator Cl-channel activity. J. Biol. Chem. 2005, 280, 5221-5226. in the first cytoplasmic loop (H139R or G149R) prevents the establishment of these interactions. Login to comment