ABCMdb

PMID: 15537638

Choi MY, Partridge AW, Daniels C, Du K, Lukacs GL, Deber CM
Destabilization of the transmembrane domain induces misfolding in a phenotypic mutant of cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2005 Feb 11;280(6):4968-74. Epub 2004 Nov 10., 2005-02-11 [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Arg347Pro ABCC7 p.Leu346Pro ABCC7 p.Leu346Pro Destabilization of the Transmembrane Domain Induces Misfolding in a Phenotypic Mutant of Cystic Fibrosis Transmembrane Conductance Regulator* Received for publication, September 1, 2004, and in revised form, November 8, 2004 Published, JBC Papers in Press, November 10, 2004, DOI 10.1074/jbc.M410069200 Mei Y. Choi‡§¶, Anthony W. Partridge‡§ʈ, Craig Daniels**‡‡, Kai Du**‡‡, Gergely L. Lukacs**‡‡§§, and Charles M. Deber‡§ §§ From the ‡Division of Structural Biology and Biochemistry and **Program in Cell and Lung Biology, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8 and the Departments of §Biochemistry and ‡‡Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada Two phenotypic missense mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel pore (L346P and R347P in transmembrane (TM) segment 6) involve gain of a proline residue, but only L346P represents a significant loss of segment hydropathy. Login to comment 1 ABCC7 p.Arg347Pro ABCC7 p.Leu346Pro We show here that, for synthetic peptides corresponding to sequences of CFTR TM6 segments, circular dichroism spectra of wild type and R347P TM6 in membrane mimetic environments are virtually identical, but L346P loses ϳ50% helicity, implying a membrane insertion defect in the latter mutant. Login to comment 2 ABCC7 p.Leu346Pro A similar defect was observed in the corresponding double-spanning ("hairpin") TM5/6-L346P synthetic peptide. Login to comment 3 ABCC7 p.Arg347Pro ABCC7 p.Leu346Pro Examination of the biogenesis of CFTR revealed that the full-length protein harboring the L346P mutation is rapidly degraded at the endoplasmic reticulum (ER), whereas the wild type and the R347P protein process normally. Login to comment 4 ABCC7 p.Leu346Pro ABCC7 p.Leu346Pro ABCC7 p.Arg347Ile Furthermore, a second site mutation (R347I) that restores in vitro membrane insertion and folding of the TM5/6-L346P peptide also rescues the folding and cell surface chloride channel function of full-length L346P CFTR. Login to comment 15 ABCC7 p.Arg347Pro ABCC7 p.Leu346Pro A number of mutations occurring in TM5/6 have been found to cause mild (usually pancreatic sufficient) forms of CF, two of which involve introduction of a proline residue: L346P, a mutation that was identified in two unrelated Cypriot patients in 1994 (10); and a second sequentially adjacent CF-phenotypic mutant, R347P (11). Login to comment 35 ABCC7 p.Leu346Pro Furthermore, in CFTR mutant L346P, the loss of Leu significantly increases the local hydrophilicity of this TM segment, i.e. on the Liu-Deber hydropathy index where values are scaled between ϩ5 and -5, Leu ranks third (ϩ4.76), whereas Pro ranks 19th (-4.92) out of the 20 commonly occurring amino acids (19). Login to comment 36 ABCC7 p.Arg347Pro In the case of R347P, the Arg positive charge is lost, but this mutation exchanges a polar residue (Arg ranks 14th, at -2.77) with one of comparable hydrophilicity. Login to comment 37 ABCC7 p.Arg347Pro ABCC7 p.Leu346Pro In the present work, we have used solid-phase peptide synthesis to prepare sequences corresponding to TM6 segments of wild type (WT) and mutants L346P and R347P of CFTR, along with some corresponding double-spanning TM5/6 peptides for comparative structural analyses. Login to comment 38 ABCC7 p.Arg347Pro ABCC7 p.Leu346Pro In parallel, we examined the relative effects of L346P versus R347P on cellular processing of full-length CFTR. Login to comment 58 ABCC7 p.Arg347Pro ABCC7 p.Arg347His ABCC7 p.Leu346Pro Construction and Expression of CFTR Variants in Mammalian Cells-The L346P, R347P, and R347H CFTR mutants were constructed FIG. 1. Login to comment 60 ABCC7 p.Arg347Pro ABCC7 p.Leu346Pro ABCC7 p.Leu346Pro ABCC7 p.Arg347Ile A, wild type sequence; B, TM5/6-L346P; C, TM5/6-R347P; and D, TM5/6-L346P-R347I. Login to comment 70 ABCC7 p.Arg347Pro ABCC7 p.Leu346Pro Baby hamster kidney (BHK) cells were stably transfected with the pNUT expression plasmids, containing the wild type (WT), L346P, or R347P CFTR, harboring an HA-epitope in the C-terminal tail of CFTR (CFTR-CintHA) (21). Login to comment 84 ABCC7 p.Arg347Pro ABCC7 p.Leu346Pro RESULTS Hydrophobicity Threshold of CF-phenotypic Mutant TM Segments-Although both L346P and R347P represent a gain of a Pro residue in CFTR TM6, the resulting local 346/347 diads (PR and LP, respectively) differ significantly in hydrophobic character. Login to comment 86 ABCC7 p.Leu346Pro ABCC7 p.Leu346Pro The outputs for the wild type (WT) and L346P CFTR sequences support an initial hypothesis that the L346P mutation decreases the average net hydrophobicity of the original TM6 segment sufficiently to prevent the proper membrane insertion of the full TM6 segment (Fig. 1, A and B). Login to comment 88 ABCC7 p.Leu346Pro In contrast, the output for the L346P mutant sequence (residues 330-341) fails to predict a sufficiently long stretch of amino acids that are above the threshold hydrophobicity required for a TM helix (27, 28). Login to comment 89 ABCC7 p.Arg347Pro ABCC7 p.Arg347Pro ABCC7 p.Leu346Pro However, unlike L346P, the R347P mutation doesn`t involve a significant change in hydrophobicity, and TM Finder predicts that the R347P mutant has the same membrane-inserted amino acid stretch (residues 330-349) as the WT TM6 sequence (Fig. 1C). Login to comment 93 ABCC7 p.Arg347Pro ABCC7 p.Leu346Pro ABCC7 p.Leu346Pro A, CD spectra of TM6-WT, TM6-L346P, and TM6-R347P peptides in lysophosphatidylcholine (LPC) detergent micelles. B, CD spectra for TM5/ 6-WT and TM5/6-L346P peptides in LPC micelles. Login to comment 94 ABCC7 p.Leu346Pro The TM5/6-L346P displays a decrease in the helical content when compared with the TM5/6-WT spectrum. Login to comment 95 ABCC7 p.Phe342Trp ABCC7 p.Phe342Trp C, Trp fluorescence spectra for TM6-WT(F342W) and TM6-L346P(F342W) peptides in LPC micelles. Login to comment 97 ABCC7 p.Leu346Pro D, Trp fluorescence spectra for the unlabeled and labeled TM5/6 and L346P peptides in LPC micelles. FRET measurements were performed using peptides labeled with dansyl chloride as the acceptor fluorophore with the Trp residue in TM6 serving as a donor fluorophore. Login to comment 99 ABCC7 p.Arg347Pro ABCC7 p.Leu346Pro synthesized Lys-tagged versions of the TM6-WT and the two mutant (L346P and R347P) sequences (Table I). Login to comment 102 ABCC7 p.Leu346Pro Although the TM6-WT peptide adopted an ␣-helical structure in the presence of LPC micelles, the TM6-L346P peptide displays only ϳ50% of the helicity observed for the TM6-WT sequence. Login to comment 103 ABCC7 p.Arg347Pro In contrast, the CD spectra of TM6-R347P and the TM6-WT peptides are virtually superimposable. Login to comment 104 ABCC7 p.Leu346Pro ABCC7 p.Leu346Pro Because the L346 locus appeared to be most affected by the Pro mutation, we further examined the effect of the L346P mutation in an expanded context by synthesizing the helix-loop-helix TM5/6-WT and TM5/6-L346P peptides. Login to comment 106 ABCC7 p.Leu346Pro CD spectra indicate that the TM5/6-L346P construct exhibits a 25% decrease in helicity when compared with the TM5/6-WT construct in SDS micelles, consistent with a 50% decrease in one TM helix (Fig. 2B). Login to comment 107 ABCC7 p.Leu346Pro Fluorescence Studies of CFTR Single TM6 and Double TM5/6 Peptides-The proposition that the L346P TM segment is only partially inserted into micellar membranes was further examined by fluorescence experiments. Login to comment 108 ABCC7 p.Phe342Trp ABCC7 p.Phe342Trp ABCC7 p.Phe342Trp To this end, we synthesized two additional peptides containing a TM-embedded fluorescent probe introduced through the conservative F342W mutation (TM6-WT(F342W) and TM6-L346P(F342W), respectively) (Table I). Login to comment 109 ABCC7 p.Leu346Pro Characteristic Trp fluorescence spectra of these two peptides in detergent micelles are presented in Fig. 2C. Noting that a membrane-embedded Trp residue will typically display increased fluorescence intensity with a blue-shifted position versus an aqueous-located counterpart, the data indicate that the Trp residue in the WT species resides in an apolar environment (maximum near 320 nm) whereas the Trp in the L346P peptide is largely aqueous exposed (shoulder near 340 nm), supporting the notion that the apolar-to-polar mutation prevents proper TM6 insertion. Login to comment 116 ABCC7 p.Arg347Pro ABCC7 p.Arg347His ABCC7 p.Leu346Pro ABCC7 p.Leu346Pro ABCC7 p.Arg347Ile WT, L346P, R347P, L346P/R347I, and R347H CFTR expression was assayed by immunoblotting, using the mouse monoclonal anti-HA Ab. Login to comment 120 ABCC7 p.Leu346Pro C, biosynthetic processing of the WT and L346P CFTR was monitored in stably transfected BHK cells by the metabolic pulse-chase technique as described under "Experimental Procedures." Login to comment 124 ABCC7 p.Leu346Pro D, relative translational rate of the full-length L346P CFTR. Login to comment 126 ABCC7 p.Leu346Pro To avoid clonal variations, COS-1 cells were transiently transfected with the WT and L346P CFTR. Login to comment 129 ABCC7 p.Leu346Pro The radioactivity associated with the WT and L346P CFTR was normalized for cellular protein and was not more than 4% variant in two experiments. Login to comment 133 ABCC7 p.Leu346Pro The FRET results suggest that (i) the WT sequence is likely folded into a helical hairpin and (ii) a substantial increase in fluorophore separation occurs between the N and C termini of the L346P mutant hairpin compared with the WT sequence. Login to comment 134 ABCC7 p.Leu346Pro The L346P Mutation Impairs the Folding of CFTR in Vivo- ER-retained, core-glycosylated (or incompletely folded) CFTR can be readily distinguished from the mature, complex-glycosylated (or folded) CFTR by immunoblot analysis, based on the faster electrophoretic mobility of the core-glycosylated form compared with the complex-glycosylated CFTR. Login to comment 135 ABCC7 p.Arg347Pro ABCC7 p.Leu346Pro Because impaired post-translational folding of the CFTR usually causes its biosynthetic processing arrest, we examined the processing of full-length L346P- and R347P-CFTR by immunoblotting and pulse-chase analysis of BHK cells, which stably express CFTR. Login to comment 137 ABCC7 p.Arg347Pro ABCC7 p.Leu346Pro As shown in Fig. 3A, immunoblot analysis of equal amounts of cell extracts demonstrated that the L346P, but not the R347P, mutation, prevented the expression of the complex-glycosylated CFTR. Login to comment 138 ABCC7 p.Arg347His The R347H missense mutation, associated with a mild functional defect of CFTR channel activity, was also expressed at the same level as the WT CFTR, confirming previous reports (31). Login to comment 139 ABCC7 p.Leu346Pro Similar results were obtained in transiently transfected COS-1 cells, indicating that the cellular phenotype of the L346P CFTR is independent of the expression system used (Fig. 3B). Login to comment 140 ABCC7 p.Leu346Pro Impaired steady-state expression of L346P-CFTR could be a consequence of its rapid degradation at the ER and/or of accelerated removal of the channel from post-Golgi compartments (32). Login to comment 141 ABCC7 p.Leu346Pro To distinguish between these scenarios, the biogenesis of L346P CFTR was monitored in BHK cells by the metabolic pulse-chase technique. Login to comment 142 ABCC7 p.Leu346Pro Although the accumulation of the complex-glycosylated WT-CFTR was obvious after 2 h of chase, the L346P mutation prevented the appearance of the complex-glycosylated form as shown by autoradiography (Fig. 3C). Login to comment 143 ABCC7 p.Leu346Pro These results suggest that the L346P mutation imposes a folding defect on CFTR, leading to the retention and degradation of the mutant at the ER. Login to comment 144 ABCC7 p.Leu346Pro ABCC7 p.Leu346Pro On the other hand, the L346P mutation does not appear to cause premature translational termination or failure of the TM5-6 or TM7-8 segments to insert into the ER, because similar amounts of radioactively labeled core-glycosylated WT and L346P CFTR were accumulated during a 10-min radioactive labeling (Fig. 3D). Login to comment 145 ABCC7 p.Leu346Pro A Second Site Mutation in TM6 Restores Biosynthetic Processing of CFTR-To test the assumption that destabilization of local TM5/6 hairpin formation may inhibit post-translational folding in the context of full-length CFTR, we searched for a second site mutation that could re-establish the stability of the L346P TM5/6. Login to comment 146 ABCC7 p.Leu346Pro ABCC7 p.Leu346Pro ABCC7 p.Arg347Ile Consideration of amino acid replacements in the vicinity of the L346P mutation identified a second site mutation (R347I) that restored the hydrophobicity of the TM6 L346P-containing segment to the threshold level that ensured membrane insertion according to TM Finder (Fig. 1D) (26). Login to comment 147 ABCC7 p.Leu346Pro ABCC7 p.Arg347Ile The L346P/R347I single spanning TM6 peptide was first synthesized, and analysis of its CD spectrum confirmed that this mutation restored the ␣-helical content of this TM6 double mutant to its WT counterpart (Fig. 4A). Login to comment 148 ABCC7 p.Leu346Pro ABCC7 p.Arg347Ile We then assessed whether the R347I mutation could restore hairpin formation of the TM5/6-L346P polypeptide by the FRET assay. Login to comment 149 ABCC7 p.Leu346Pro ABCC7 p.Arg347Ile Using a TM5/6-L346P/R347I peptide in which a Trp residue was inserted near the C terminus (Table I), and in which the N terminus was labeled with a dansyl group, we found that the donor fluorescence quench in the double mutant was now similar to that of the WT TM5/6 (Fig. 4B). Login to comment 150 ABCC7 p.Leu346Pro If destabilization of the TM5/6 hairpin accounts for the processing defect of the L346P CFTR, introducing the second site mutation should correspondingly restore the folding and biosynthetic processing of full-length CFTR. Login to comment 152 ABCC7 p.Leu346Pro ABCC7 p.Leu346Pro ABCC7 p.Arg347Ile Immunoblot analysis demonstrated the appearance of the complex-glycosylated L346P/R347I CFTR in both transiently transfected COS-1 and stably transfected BHK cells, whereas no detectable amount of L346P CFTR was present (Figs. Login to comment 154 ABCC7 p.Leu346Pro Functional assessment of the plasma membrane protein kinase A-activated halide conductance confirmed the partial reversion of the processing defect by demonstrating that the cAMP-stimulated iodide release of the L346P CFTR (6.3 Ϯ 0.2 nmol/min) was increased by 3-fold in the presence of the second site mutation (18.4 Ϯ 03 nmol/min) (Fig. 5B). Login to comment 155 ABCC7 p.Leu346Pro The detection of L346P CFTR by functional assay, but not by immunoblotting, is conceivable due to the higher sensitivity of the iodide efflux assay. Login to comment 160 ABCC7 p.Leu346Pro ABCC7 p.Leu346Pro ABCC7 p.Arg347Ile ABCC7 p.Arg347Ile A, CD spectra of TM6-WT and TM6-L346P/R347I in LPC micelles. B, Trp fluorescence spectra for the unlabeled and labeled TM5/ 6-WT and TM5/6-L346P/R347I peptides in LPC micelles. FRET measurements were performed using peptides labeled with dansyl chloride as the acceptor fluorophore with the Trp residue in TM6 serving as a donor fluorophore. Login to comment 164 ABCC7 p.Arg347Pro ABCC7 p.Leu346Pro ABCC7 p.Leu346Pro Thus, despite the similarity of the two mutations and their adjacent positions in the sequence, the TM6-L346P peptide displayed only ϳ50% of the helicity observed for the TM6-WT sequence, whereas the TM6-R347P retained WT character, indicating that the ability of the L346P peptide to properly insert into the apolar milieu has been significantly compromised (Fig. 2A). Login to comment 167 ABCC7 p.Leu346Pro To address this situation, we synthesized and compared helix-loop-helix peptides corresponding to the CFTR TM5/6-WT and the TM5/6-L346P sequence. Login to comment 169 ABCC7 p.Leu346Pro ABCC7 p.Leu346Pro Fluorescence resonance energy transfer experiments on labeled TM5/6 constructs further suggested that L346P prevents formation of proper TM6 topology, because FRET effects were significantly reduced in the TM5/ 6-L346P peptide versus the corresponding WT construct. Login to comment 176 ABCC7 p.Leu346Pro Thus, a mutation in TM6 such as L346P may further reduce the efficiency of proper membrane anchoring of the protein. Login to comment 177 ABCC7 p.Leu346Pro Consistent with these considerations, we found that full-length CFTR protein harboring the L346P mutation is subjected to core glycosylation but was unable to fold and was rapidly degraded in vivo. Login to comment 179 ABCC7 p.Leu346Pro Because the full-length L346P protein is indeed synthesized, an additional possibility is that the protein is able to compensate, at least in part, for the topological defect at the TM5/6 locus via TM-packing interactions with the second (TM7-12) CFTR TM domain. Login to comment 180 ABCC7 p.Leu346Pro Based on our study, it appears that L346P may affect local CFTR TM5/6 structure to such an extent that the ER-associated quality-control mechanism recognizes the mutant as non-native and marks it for degradation. Login to comment 181 ABCC7 p.Leu346Pro As a result, escape from the ER and cell surface delivery of L346P CFTR is severely compromised FIG. 5. Login to comment 182 ABCC7 p.Leu346Pro The effect of a second site mutation on the expression and function of L346P CFTR. Login to comment 186 ABCC7 p.Leu346Pro ABCC7 p.Leu346Pro ABCC7 p.Arg347Ile B, cAMP-stimulated iodide efflux of BHK cells expressing wild-type (wt), L346P, or L346P/ R347I CFTR. Login to comment 190 ABCC7 p.Leu346Pro Schematic model of the possible topological consequences of the L346P mutation in CFTR. Login to comment 200 ABCC7 p.Leu346Pro ABCC7 p.Arg347Ile The partial reversion of the L346P CFTR processing defect by a second site mutation (R347I) (Fig. 3B), which restores full-length TM6 insertion potential (Fig. 1D), suggests that segment hydrophobicity is prominent among the factors that play determining roles in the post-translational folding of CFTR. Login to comment 203 ABCC7 p.Leu346Pro Because the resulting TM6 is now too short to span the cellular bilayers, a compensatory "pull" on the residues within the TM5/6-L346P loop region may also occur (Fig. 6B). Login to comment 208 ABCC7 p.Leu346Pro Based on our results, impaired post-translational folding of CFTR is the primary defect in the case of the L346P mutation. Login to comment 209 ABCC7 p.Arg347Pro In contrast, the effects of the R347P mutation present an alternate scenario. Login to comment 211 ABCC7 p.Arg347Pro Thus, the loss of a WT interhelical salt bridge (12) in R347P may contribute to the underlying defect. Login to comment 212 ABCC7 p.Leu346Pro ABCC7 p.Arg347Ile Note that this salt bridge would similarly be abolished in the L346P/R347I mutant, perhaps explaining, in part, why this "rescue mutant" is not fully functional. Login to comment 215 ABCC7 p.Arg347Pro However, loss of an Arg residue at the adjacent position appears to induce a more downstream event, viz., R347P may promote changes in the selectivity or effectiveness of the channel pore of CFTR stemming from loss of side-chain positive character (43-46), rather than preventing post-translational folding. Login to comment