ABCMdb

PMID: 20032308

Roy G, Chalfin EM, Saxena A, Wang X
Interplay between ER exit code and domain conformation in CFTR misprocessing and rescue.
Mol Biol Cell. 2010 Feb 15;21(4):597-609. Epub 2009 Dec 23., 2010-02-15 [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC7 p.Arg555Lys R555K, a mutation that rescues ⌬F508 misprocessing, improves Sec24 association and enhances its post-ER stability. Login to comment 7 ABCC7 p.Arg555Lys Using in situ limited proteolysis, we demonstrated a clear change in trypsin sensitivity in ⌬F508 NBD1, which is reversed, together with that of other domains, by low temperature, R555K or both. Login to comment 9 ABCC7 p.Arg555Lys Low temperature and R555K are additive in improving ⌬F508 conformational maturation and processing. Login to comment 50 ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys The pcDNA3.1(ϩ)-CFTR-⌬F508/R555K and pcDNA3.1(ϩ)- CFTR-⌬F508/DAA were constructed by introducing R555K and DAA, respectively, into pcDNA3.1(ϩ)-CFTR-⌬F508. Login to comment 51 ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys The pcDNA3.1(ϩ)-CFTR-⌬F508/ R555K/DAA was constructed by introducing DAA into pcDNA3.1(ϩ)-CFTR- ⌬F508/R555K. Login to comment 52 ABCC7 p.Arg29Lys ABCC7 p.Arg29Lys To construct pcDNA3.1(ϩ)-CFTR-⌬F508/R29K, an NdeI-BspeI fragment including the 5Ј untranslated region, the amino terminal tail (NT) and MSD1 of CFTR was used as a cassette, and R29K substitution was introduced into pcDNA3.1(ϩ)-CFTR-⌬F508 using this cassette. Login to comment 53 ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys ABCC7 p.Arg29Lys ABCC7 p.Arg29Lys Finally, the pcDNA3.1(ϩ)- CFTR-⌬F508/R29K/R555K was constructed by replacing the BspeI-HpaI fragment of pcDNA3.1(ϩ)-CFTR-⌬F508/R29K with the corresponding fragment from pcDNA3.1(ϩ)-CFTR-⌬F508/R555K. Login to comment 65 ABCC7 p.Arg555Lys HEK293 cells stably expressing DAA, ⌬F508/DAA and ⌬F508/R555K CFTR were generated by transfecting HEK293 cells with specific CFTR expression plasmids and selecting in the presence of 400 ␮g/ml G418 (A. Login to comment 68 ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys BHK cells stably expressing ⌬F508/R555K CFTR (BHK-⌬F/R555K) were generated by transfecting BHK cells with the CFTR expression plasmid and selecting with 400 ␮g/ml G418. Login to comment 207 ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys ABCC7 p.Arg29Lys R555K Rescues ⌬F508 CFTR by Improving Both Export and Post-ER Stability R555K, alone (Teem et al., 1996) or in combination with R29K (Hegedus et al., 2006) rescues ⌬F508 CFTR. Login to comment 208 ABCC7 p.Arg555Lys ABCC7 p.Arg29Lys We found that R29K does not improve ⌬F508 processing, nor does it contribute to ⌬F508 rescue when combined with R555K (2RK) in HEK293 cells and this is true at both 37 and 30°C (Figure 7A). Login to comment 209 ABCC7 p.Arg555Lys ABCC7 p.Arg29Lys In BHK cells, R29K alone inhibits ⌬F508 processing but only slightly enhances ⌬F508 processing when combined with R555K at 37°C (Supplemental Figure 2). Login to comment 210 ABCC7 p.Arg555Lys Of note, in HEK293 cells, the combination of low temperature and R555K additively increases the processing of ⌬F508 based on the percent of total band C (Figure 7A), whereas the enhancement in processing by the combination of the two is much less in BHK cells (Supplemental Figure 2). Login to comment 211 ABCC7 p.Arg555Lys Consistent with the role of the "DAD" motif as the ER exit code in the context of ⌬F508 CFTR, the enhanced processing of ⌬F508 CFTR by introducing R555K at both 37 and 30°C is abolished by further introduction of DAA mutation (Figure 7B). Login to comment 212 ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys To probe the mechanism of R555K rescue of ⌬F508 CFTR, we first observed the kinetic accumulation of ⌬F508/R555K in bands B and C and compared it with that of ⌬F508. Login to comment 213 ABCC7 p.Arg555Lys Although the rates of accumulation in band B are largely similar between the two forms of CFTR, R555K significantly increases the accumulation of ⌬F508 CFTR in band C (Figure 7C), suggesting increased export and/or post-ER stability. Login to comment 214 ABCC7 p.Arg555Lys CHX chase indicated a moderate increase in the post-ER stability by R555K (Figure 7D), and CFTR quantitative co-immunoprecipitation revealed an increase in association with Sec24 (Figure 7E). Login to comment 215 ABCC7 p.Arg555Lys These results suggest that R555K rescues ⌬F508 CFTR through enhancing both export and post-ER stability. Login to comment 216 ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys Rescue of ⌬F508 CFTR by Low Temperature or R555K Is Accompanied by the Enhancement in Global Conformational Maturation We probed domain conformation of ⌬F508 CFTR after rescue with low temperature, R555K, or both using in situ limited proteolysis. Login to comment 220 ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys The introduction of R555K has a similar effect (Figure 8Ac), suggesting that R555K substitution alters the folding of ⌬F508 CFTR in a way that achieves a comparable level of conformational maturation as the low-temperature rescue. Login to comment 221 ABCC7 p.Arg555Lys Although "⌬F508, 30°C", "⌬F508/R555K," and "DAA" have comparable distribution between bands B and C, at the steady state, the relative intensity of the 37-kDa band is higher and the 42-kDa fragments are more resistant to tryp- Figure 5. Login to comment 233 ABCC7 p.Arg555Lys Notably, when low temperature is combined with R555K in HEK293 cells, we have observed a dramatic increase in the trypsin resistance of the 42-kDa fragments and an increased relative intensity of the 37-kDa band, which together lead to a tryptic pattern similar to wild-type CFTR (Figure 8A, d and e). Login to comment 238 ABCC7 p.Arg555Lys In contrast, R555K substitution results in a more uniform tryptic pattern (Figure 8Ah). Login to comment 239 ABCC7 p.Arg555Lys Although the 30-kDa fragment typical of wild-type conformation is now clearly present (black arrowhead), the 34-kDa common band that is present in ⌬F508, DAA and wild-type CFTR is greatly diminished (Figure 8A, f-h, gray arrowheads), suggesting that R555K produces an additional conformational change in NBD2. Login to comment 240 ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys The combination of low temperature and R555K did not produce additional conformational correction in NBD2 over what has been achieved by R555K alone (Figure 8A, h and i). Login to comment 241 ABCC7 p.Arg555Lys Taken together, we show that rescue of ⌬F508 by low temperature or R555K is associated with improved conformational maturation in multiple domains. Login to comment 266 ABCC7 p.Arg555Lys To test the relationship between ⌬F508 rescue and its domain conformation, we conducted in situ limited proteolysis on microsomes prepared from BHK-WT cells, BHK-⌬F cells incubated at 37 or 30°C, and BHK-⌬F/R555K cells. Login to comment 271 ABCC7 p.Arg555Lys In contrast, BHK cells stably expressing ⌬F508/R555K showed a clear appearance of the 37-kDa bands at high trypsin concentrations (Figure 9Bd, black arrowhead). Login to comment 274 ABCC7 p.Arg555Lys In contrast, R555K was able to restore the 50-kDa pattern to wild-type level. Login to comment 277 ABCC7 p.Arg555Lys Parallel to the behavior of the amino terminal module, reducing temperature did not lead to the appearance of the 30-kDa fragment (Figure 9Bg) but R555K did (Figure 9Bh). Login to comment 284 ABCC7 p.Arg555Lys R555K improves export and post-ER stability of ⌬F508 CFTR. Login to comment 285 ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys ABCC7 p.Arg29Lys ABCC7 p.Arg29Lys (A) HEK293 cells were transiently transfected with ⌬F, ⌬F/ R29K, ⌬F/R555K and ⌬F/R29K/R555K (⌬F/ 2RK) and cultured at 37°C for 20 h (37°C), or further switched to 30°C and incubated for an additional 16 h (30°). Login to comment 290 ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys (B) HEK293 cells were transiently transfected with ⌬F, ⌬F/R555K and ⌬F/ R555K/DAA CFTR. Login to comment 295 ABCC7 p.Arg555Lys (C) HEK293 cells were transiently transfected with ⌬F or ⌬F/ R555K CFTR and incubated at 37°C for the indicated time. Login to comment 300 ABCC7 p.Arg555Lys (D) CHX chase on HEK293 cells stably expressing ⌬F and ⌬F/R555K was performed and quantified as described in Figure 2. Login to comment 301 ABCC7 p.Arg555Lys (E) HEK293 cells were transiently transfected with ⌬F or ⌬F/R555K. Login to comment 318 ABCC7 p.Arg555Lys 3) The pattern of ⌬F508 CFTR reverts to wild-type-like upon rescue by low temperature or R555K or both in HEK293 cells (Figure 8). Login to comment 326 ABCC7 p.Arg555Lys Low temperature and R555K promote the conformational maturation of ⌬F508 CFTR in HEK293 cells. Login to comment 328 ABCC7 p.Arg555Lys For ⌬F, 30°C and ⌬F/R555K, 30°C, cells were incubated at 30°C for 16 h before the preparation of microsomes. Login to comment 333 ABCC7 p.Arg555Lys ⌬F508 CFTR has reduced coupling to Sec24, which is reversed by low temperature (Wang et al., 2004, 2008) or R555K (Figure 7E). Login to comment 337 ABCC7 p.Arg555Lys ABCC7 p.Arg29Lys We found that R29K does not contribute to ⌬F508 rescue in HEK 293 cells (Figure 7A) but slightly improves ⌬F508 rescue only in combination with R555K in BHK cells (Supplemental Figure 2). Login to comment 338 ABCC7 p.Arg555Lys R555K rescues ⌬F508 CFTR by improving its coupling to COPII and moderately increasing its post-ER stability (Figure 7, D and E). Login to comment 340 ABCC7 p.Arg553Met Interestingly, R553M, the substitution of the other arginine in the same RXR motif, rescues ⌬F508 CFTR as well (Teem et al., 1993), but through enhancing the folding yield of ⌬F508 NBD1 based on an in vitro study (Qu et al., 1997). Login to comment 343 ABCC7 p.Arg553Met ABCC7 p.Arg555Lys Instead, like other second site mutations in NBD1, R555K and R553M substitutions might alter the conformation of CFTR in a way that repairs ⌬F508 conformation defects. Login to comment 344 ABCC7 p.Arg553Met ABCC7 p.Arg555Lys The ER exit code "DAD", F508, and many ⌬F508 rescuing mutations (including R555K and R553M) reside in NBD1. Login to comment 346 ABCC7 p.Arg555Lys This is partly attributable to the inclusion of multiple "solubilization mutations" into ⌬F508 NBD1 to facilitate its purification (Lewis et al., 2005), several of which, including R555K, rescue the ⌬F508 export and channel gating defects (Pissarra et al., 2008). Login to comment 361 ABCC7 p.Arg555Lys Although ⌬F508 mutation initiates a global conformational change from NBD1, R555K, originating in same domain, reverses the global conformational defect. Login to comment 363 ABCC7 p.Arg555Lys Reducing the temperature produces a similar effect on ⌬F508 CFTR conformation as R555K in HEK293 cells (Figure 8). Login to comment 377 ABCC7 p.Arg555Lys Rescue maneuvers such as low temperature and R555K restore both its ER export (Figure 7E; Wang et al., 2004) and post-ER stability (Figure 7D; Sharma et al., 2001; Varga et al., 2008), and these processes are highly dependent upon the ER exit code "DAD" (Figures 6 and 7B). Login to comment 381 ABCC7 p.Arg555Lys R555K improves the ER export (Figure 7E), post-ER stability (Figure 7D), and channel gating (Teem et al., 1996) of ⌬F508 CFTR. Login to comment 386 ABCC7 p.Arg555Lys Combining R555K with low-temperature additively improves the conformational maturation of ⌬F508 CFTR (Figure 8) and its processing (Figure 7A) in HEK293 cells. Login to comment