ABCMdb

PMID: 10362539

Ostedgaard LS, Zeiher B, Welsh MJ
Processing of CFTR bearing the P574H mutation differs from wild-type and deltaF508-CFTR.
J Cell Sci. 1999 Jul;112 ( Pt 13):2091-8., [PubMed]

Sentences

No. Mutations Sentence Comment

18 ABCC7 p.Pro574His ABCC7 p.Ala455Glu Earlier studies showed that the CF-associated mutants, P574H and A455E, were also misprocessed. Login to comment 19 ABCC7 p.Pro574His ABCC7 p.Ala455Glu In this study, we found that processing of P574H and A455E was also temperature-sensitive; at 26°C, some of the protein matured. Login to comment 20 ABCC7 p.Pro574His In contrast to other CFTR mutants, P574H accumulated in punctate cytoplasmic bodies that colocalized with endoplasmic reticulum (ER) markers. Login to comment 22 ABCC7 p.Pro574His P574H showed a prolonged association with Hsp70 and also colocalized with Hsp70. Login to comment 24 ABCC7 p.Pro574His Unlike wild-type CFTR, which was converted into an intermediate that was stable in the presence of BFA at 37°C, ∆F508 and P574H produced the intermediate only when the temperature was reduced to 26°C. Furthermore the wild-type intermediate was not associated with Hsp70. Login to comment 26 ABCC7 p.Pro574His Key words: Cystic fibrosis, Hsp70, Protein biosynthesis SUMMARY Processing of CFTR bearing the P574H mutation differs from wild-type and ∆F508-CFTR Lynda S. Ostedgaard, Bernhardt Zeiher and Michael J. Welsh* Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA *Author for correspondence Accepted 22 April; published on WWW 10 June 1999 in NBD1 and throughout the protein (Tsui, 1995). Login to comment 27 ABCC7 p.Pro574His ABCC7 p.Ala455Glu We earlier studied two other CF-associated mutations located in NBD1, A455E and P574H (Sheppard et al., 1995). Login to comment 29 ABCC7 p.Pro574His ABCC7 p.Ala455Glu However, A455E and P574H generate reduced net epithelial current because the proteins are misprocessed and few functional channels reach the plasma membrane (Sheppard et al., 1995; Champigny et al., 1995). Login to comment 30 ABCC7 p.Pro574His ABCC7 p.Ala455Glu Nevertheless, the processing defect of A455E and P574H is less pronounced than that of ∆F508 and the resulting clinical phenotype is less severe (Kristidis et al., 1992; Kerem et al., 1990a; Veeze et al., 1994; Gan et al., 1995). Login to comment 32 ABCC7 p.Pro574His ABCC7 p.Ala455Glu In this study, we compared the processing of P574H and A455E mutants to that of ∆F508. Login to comment 33 ABCC7 p.Pro574His By studying mutants with different degrees of misprocessing, we hope to gain further insight into the biosynthesis of both normal and mutant CFTR which may help design interventions to improve the processing of ∆F508, P574H, and possibly other CF-associated mutants. Login to comment 37 ABCC7 p.Pro574His ABCC7 p.Ala455Glu Using the pcDNA3-6His-CFTR as a backbone, we made the constructs A455E, P574H and ∆F508 (Kunkel, 1985) and confirmed the mutations by DNA sequencing in both directions. Login to comment 65 ABCC7 p.Pro574His ABCC7 p.Ala455Glu RESULTS Because earlier studies suggested that lowering the temperature allowed ∆F508 to fold correctly and exit the ER (Denning et al., 1992a; Lukacs et al., 1993; Sato et al., 1996), we first examined the temperature-sensitivity of A455E and P574H processing. Login to comment 70 ABCC7 p.Pro574His ABCC7 p.Ala455Glu For the NBD1 mutants, ∆F508 (B), A455E (C) and P574H (D), band B was the primary form detected at 37°C. Login to comment 74 ABCC7 p.Pro574His ABCC7 p.Ala455Glu For ∆F508 (B) and A455E (C), the relative amount of band C was minimal at each time at 37°C. Although the relative amount of band C in P574H (D) was also low, it increased slowly with time at 37°C. Login to comment 75 ABCC7 p.Pro574His ABCC7 p.Pro574His ABCC7 p.Ala455Glu When the temperature was reduced to 26°C, the relative amount of band C for ∆F508 and A455E increased modestly, while the amount of P574H band C relative to total P574H increased. Login to comment 76 ABCC7 p.Pro574His ABCC7 p.Pro574His Although lowering the temperature caused an increase in the relative amount of P574H band C, the total amount of P574H band C was not as high as that in wild-type CFTR. Login to comment 77 ABCC7 p.Pro574His ABCC7 p.Ala455Glu These results indicate that, like ∆F508, both A455E and P574H are temperature-sensitive processing mutants. Login to comment 78 ABCC7 p.Pro574His ABCC7 p.Ala455Glu Moreover, P574H makes relatively more band C at both 37°C and 26°C than either ∆F508 or A455E. Login to comment 82 ABCC7 p.Pro574His ABCC7 p.Ala455Glu P574H and A455E are temperature-sensitive mutants. Login to comment 83 ABCC7 p.Pro574His ABCC7 p.Ala455Glu COS-7 cells were electroporated with pcDNA3 vectors encoding wild-type CFTR (A), ∆F508 (B), A455E (C), and P574H (D). Login to comment 92 ABCC7 p.Pro574His ABCC7 p.Pro574His ABCC7 p.Pro574His ABCC7 p.Pro574His 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 5 10 15 20 25 30 Chase (hrs) P574H - BFA P574H + BFA ∆F508 - BFA ∆F508 + BFA Wild-type - BFA Wild-type + BFA 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 5 10 15 20 25 30 Chase (hrs) P574H - BFA P574H + BFA ∆F508 - BFA ∆F508 + BFA A: 37 ˚C B: 26 ˚C RelatoveamountofBandBRelatoveamountofBandB Fig. 2. Login to comment 93 ABCC7 p.Pro574His ABCC7 p.Pro574His Effect of brefeldin A on the relative amount of band B protein at 37°C or 26°C. HeLa cells infected with recombinant vaccinia virus encoding P574H-CFTR, ∆F508-CFTR, and wild-type CFTR were pulsed (P574H-CFTR and ∆F508-CFTR for 30 minutes; wild-type-CFTR for 15 minutes) with [35S]methionine at 5 hours post-infection and chased for the indicated times with 10 mM cold methionine in the presence or absence of (5 µg/ml) brefeldin A (BFA). Login to comment 95 ABCC7 p.Pro574His Chase was continued for 30 hours for P574H and ∆F508 to detect the stable B form. Login to comment 99 ABCC7 p.Pro574His Repeated measures analysis with multiple means comparison using Supernova software (Abacus Concepts, Berkeley, CA) indicates that P574H + BFA is different from ∆F508 + BFA from 7.5 hours through 30 hours (P=0.013). Login to comment 100 ABCC7 p.Pro574His Wild-type immunoprecipitated with anti-CFTR antibodies (-BFA, ᭺; +BFA, ᭹); ∆F508 (-BFA, ᭝; +BFA, ᭡); P574H (-BFA, ᭛; + BFA, ᭜). Login to comment 107 ABCC7 p.Pro574His Unlike wild-type CFTR, neither ∆F508 nor P574H formed detectable intermediate B after BFA treatment at 37°C (Fig. 2A). Login to comment 108 ABCC7 p.Pro574His ABCC7 p.Pro574His However, when the temperature was lowered to 26°C, the intermediate B form of P574H was detectable after 7.5 hours of BFA treatment (Fig. 2B), a time that correlates with the production of P574H band C in the absence of BFA (Fig. 1D). Login to comment 109 ABCC7 p.Pro574His Likewise, a small amount of intermediate B accumulated after incubation of ∆F508-expresssing cells at 26°C, but this accumulation of ∆F508 was slower than the accumulation of P574H intermediate B at 26°C (Fig. 2). Login to comment 110 ABCC7 p.Pro574His ABCC7 p.Pro574His These data suggest that the temperature-sensitive maturation defect of ∆F508 and P574H occurs at or prior to generation of intermediate band B protein and that P574H responds more readily to lowered temperature than ∆F508. Login to comment 111 ABCC7 p.Pro574His ABCC7 p.Ala455Glu We used immunocytochemistry to determine if the cellular distribution of ∆F508, A455E and P574H was consistent with the quantitative biochemical differences we had observed. Login to comment 114 ABCC7 p.Ala455Glu The fluorescence pattern of ∆F508 (C) and A455E (E) at 37°C resembles the reticular pattern characteristic of ER with an absence of plasma membrane staining. Login to comment 115 ABCC7 p.Ala455Glu When the cells were incubated at 26°C, cytoplasmic staining became more diffuse and the outline of the cell membrane was occasionally detectable in ∆F508 (D) and A455E (F). Login to comment 117 ABCC7 p.Pro574His The NBD1 mutants are temperature-sensitive and P574H displays a unique cytoplasmic pattern of immunofluorescence at 37°C. Login to comment 118 ABCC7 p.Pro574His ABCC7 p.Ala455Glu Immunofluorescence in COS-7 cells electroporated with wild-type-CFTR (A,B); ∆F508 (C,D); A455E (E,F); and P574H (G,H). Login to comment 120 ABCC7 p.Pro574His The same punctate cytoplasmic bodies are present when P574H is expressed in HeLa cells at 37°C (not shown). Login to comment 121 ABCC7 p.Pro574His pattern, P574H presented an unusual immunofluorescence pattern of prominent punctate bodies distributed within the cytoplasm when cells were grown at 37°C (G). Login to comment 124 ABCC7 p.Pro574His We asked whether the cytoplasmic bodies produced by P574H colocalized with a known intracellular organelle. Login to comment 128 ABCC7 p.Pro574His Moreover, in cells stained with both anti-CFTR and anti-Golgi antibodies, the bodies which contain P574H (Fig. 4C) did not colocalize with the Golgi markers, p58 (Bloom and Brashear, 1989) (Fig. 4D) or β-COP (Duden et al., 1991) (not shown). Login to comment 129 ABCC7 p.Pro574His These data suggested the P574H cytoplasmic bodies were not part of the Golgi complex. Login to comment 130 ABCC7 p.Pro574His We used cAMP agonists, which have been shown by others to influence membrane insertion and retrieval of endosomal CFTR (Bradbury et al., 1992; Lehrich et al., 1998), to determine if the P574H bodies were part of endocytic or exocytic vesicles. Login to comment 132 ABCC7 p.Pro574His Although CFTR has also been reported to be contained in clathrin-coated vesicles (Bradbury et al., 1994), the punctate P574H bodies did not colocalize with the more disperse network of clathrin, a component of the trans-Golgi network and the membrane coat of endocytic vesicles and lysosomes (Brodsky, 1988) (not shown). Login to comment 133 ABCC7 p.Pro574His To determine if P574H was present in the ER, we examined the staining pattern of the ER-resident protein, protein disulfide isomerase (PDI) (Kaetzel et al., 1987). Login to comment 135 ABCC7 p.Pro574His Thus P574H, like ∆F508, is retained within the reticular network of the ER. Login to comment 136 ABCC7 p.Pro574His In addition, the punctate P574H cytoplasmic bodies stained with both anti-CFTR and anti-PDI antibodies. Login to comment 137 ABCC7 p.Pro574His ABCC7 p.Pro574His These results suggest not only that P574H is localized in the ER, but that the punctate cytoplasmic bodies may represent a subdomain of the ER which is only detectable in cells expressing P574H. Login to comment 138 ABCC7 p.Pro574His Because previous work showed that ∆F508 was associated with the cytoplasmic chaperone Hsp70 (Yang et al., 1993), we examined the possibility that the P574H in the punctate bodies might associate with Hsp70. Login to comment 139 ABCC7 p.Pro574His When the same cells are stained with both anti-CFTR and anti-Hsp70 antibodies, P574H (Fig. 5C) and Hsp70 (Fig. 5D) show a striking colocalization in both the reticular ER pattern and in the cytoplasmic bodies. Login to comment 140 ABCC7 p.Pro574His ABCC7 p.Pro574His Colocalization of P574H and Hsp70 suggested a physical association of P574H with Hsp70. Login to comment 141 ABCC7 p.Pro574His To test this, we used anti-Hsp70 antibodies to coimmunoprecipitate P574H that was bound to Hsp70 and evaluated the time course of the retention in a pulse-chase experiment. Login to comment 142 ABCC7 p.Pro574His Fig. 6 shows that band B, but not band C, of wild-type CFTR, ∆F508 and P574H were all initially associated with Hsp70. Login to comment 144 ABCC7 p.Pro574His However, band B of both ∆F508 and P574H retain their association with Hsp70 for Fig. 4. Login to comment 145 ABCC7 p.Pro574His P574H cytoplasmic bodies are not part of the Golgi complex. Login to comment 146 ABCC7 p.Pro574His COS-7 cells expressing P574H and grown at 37°C were either treated with brefeldin A (BFA) (5 µg/ml) (B) or the vehicle control (A) for 30 minutes before staining with anti-CFTR antibody. Login to comment 147 ABCC7 p.Pro574His Cells expressing P574H grown at 37°C were stained with anti-CFTR antibody (C) and anti-Golgi (p58) antibody (D). Login to comment 149 ABCC7 p.Pro574His P574H cytoplasmic bodies colocalize with ER markers and with Hsp70. Login to comment 150 ABCC7 p.Pro574His Cells expressing P574H grown at 37°C were stained with anti-CFTR antibody (A) and with protein-disulphide isomerase antibody (PDI) (B). Login to comment 151 ABCC7 p.Pro574His Cells expressing P574H grown at 37°C were stained with anti-CFTR antibody (C) and with anti-Hsp70 antibody (D). Login to comment 153 ABCC7 p.Pro574His In addition, pulse-chase studies show that relatively more of P574H is associated with Hsp70 than is wild-type CFTR (Fig. 6B). Login to comment 154 ABCC7 p.Pro574His ABCC7 p.Pro574His This continued physical association of P574H and Hsp70 is consistent with the immunocytochemical colocalization of P574H and Hsp70. Login to comment 158 ABCC7 p.Pro574His ABCC7 p.Pro574His ABCC7 p.Pro574His The continued retention of P574H with Hsp70 suggests that the conformational maturation of P574H may be delayed or actually inhibited, consistent with the diminished ability of P574H to adopt the intermediate B conformation at 37°C. Login to comment 165 ABCC7 p.Pro574His ABCC7 p.Ala455Glu DISCUSSION Earlier work showed that CFTR containing the CF-associated mutations ∆F508, A455E, or P574H decreases cell membrane Cl- current primarily because the mutant proteins fail to fold correctly and therefore do not traffic out of the ER (Cheng et al., 1990; Lukacs et al., 1994; Ward and Kopito, 1994; Sheppard et al., 1995; Qu and Thomas, 1996; Qu et al., 1997; Yang et al., 1993; Zhang et al., 1998). Login to comment 167 ABCC7 p.Pro574His The association of P574H and ∆F508 with Hsp70 is prolonged. Login to comment 168 ABCC7 p.Pro574His HeLa cells infected with recombinant vaccinia virus encoding wild-type-CFTR, ∆F508 and P574H were pulsed for 15 minutes with [35S]methionine at 5 hours post-infection and chased for the indicated times with 10 mM cold methionine at 37°C. Login to comment 174 ABCC7 p.Pro574His Wild-type-CFTR (᭺); P574H (᭛); ∆F508 (᭡). Login to comment 185 ABCC7 p.Pro574His ABCC7 p.Ala455Glu We found that, like ∆F508, the processing of P574H and A455E is temperature-sensitive. Login to comment 186 ABCC7 p.Pro574His ABCC7 p.Pro574His ABCC7 p.Ala455Glu Moreover, unlike ∆F508 and A455E, P574H formed some mature protein at 37°C, and at 26°C, P574H generated relatively more mature protein than ∆F508. Login to comment 187 ABCC7 p.Pro574His Thus the processing defect is less severe for P574H, consistent with functional studies (Sheppard et al., 1995). Login to comment 188 ABCC7 p.Pro574His These studies demonstrate that misprocessing is not an all-or-none phenomenon, but rather a continuum, with wild-type P574H >A455E>>∆F508. Login to comment 189 ABCC7 p.Pro574His ABCC7 p.Ala455Glu This is consistent with the clinical phenotype in CF patients: P574H and A455E are associated with a milder, pancreatic-sufficient phenotype and ∆F508 is associated with a severe, pancreatic-insufficient clinical phenotype (Kerem et al., 1990a,b; Kristidis et al., 1992; Veeze et al., 1994; Gan et al., 1995). Login to comment 192 ABCC7 p.Pro574His This was especially clear for the P574H mutant: the appearance of the intermediate B form in cells treated with BFA occurred at the same time as the maturation of band B to band C in the cells not treated with BFA, suggesting, as has been shown for wild-type, that intermediate B goes on to become the mature band C form of the protein. Login to comment 193 ABCC7 p.Pro574His ABCC7 p.Pro574His At 37°C, P574H resides in the ER; P574H showed both the characteristic reticular pattern of immunocytochemical staining typical of ER as well as a unique punctate pattern of cytoplasmic bodies which colocalized with an ER marker. Login to comment 194 ABCC7 p.Pro574His More interestingly, P574H also colocalized with the chaperone Hsp70 in both the reticular ER and in the punctate bodies and was associated with Hsp70 by coimmunoprecipitation. Login to comment 195 ABCC7 p.Pro574His ABCC7 p.Pro574His When the temperature was reduced to 26°C, the cytoplasmic bodies containing P574H and Hsp70 were no longer observed, suggested they had dissipated and P574H proceeded to make both the intermediate B and the band C forms of protein. Login to comment 196 ABCC7 p.Pro574His The punctate bodies appeared only in cells expressing P574H. Login to comment 197 ABCC7 p.Ala455Glu These inclusions were not present in wild-type, ∆F508, A455E or any other mutant we have studied. Login to comment 198 ABCC7 p.Pro574His This morphological pattern is not simply due to the level of protein expression, because the absolute amount of recombinant protein expressed is low and protein expression was no greater for P574H than for any other forms of CFTR. Login to comment 200 ABCC7 p.Pro574His Interestingly, the appearance of subcellular structures reminiscent of the P574H bodies have been reported following expression of other endogenous and recombinant membrane proteins. Login to comment 205 ABCC7 p.Pro574His We do not know the functional significance of the P574H accumulated within the punctate bodies; it may represent a pool of protein which can convert to the intermediate B form when the temperature is lowered. Login to comment 206 ABCC7 p.Pro574His The late onset of modest amounts of mature P574H band C at 37°C may represent a slow conversion or 'leak` from this protected pool to intermediate B. Login to comment 209 ABCC7 p.Pro574His Alternatively, we cannot exclude the possibility that the punctate bodies in cells expressing P574H could represent an ER subcompartment en-route to degradation. Login to comment 211 ABCC7 p.Pro574His ABCC7 p.Pro574His The inability of P574H to form intermediate B and the prolonged retention of P574H by Hsp70 are consistent with this observation. Login to comment