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Functional Roles of the Nucleotide-Binding Folds in The Proc. Natl. Acad. Sci. USA Vol. 90, pp. 9963-9967, November 1993 Physiology Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator (chloride channel/traffic ATPases) LISA S. SMIT*, DANIEL J. WILKINSONt, MONIQUE K. MANSOURAf, FRANCIS S. COLLINS*§1, AND DAVID C. DAWSONt Departments of *Human Genetics and tPhysiology, tBioengineering Program, and §Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109; and lNational Center for Human Genome Research, Bethesda, MD 20892 Communicated by Gerhard Giebish, July 2, 1993 ABSTRACT The cystic fibrosis transmembrane conduc- A tance regulator (CFTR), a member of the traffic ATPase NBF1 FSLLGTPVLKDINFKIERGQLLAVA STGAGKT SLLMMIMGE superfamily, possesses two putative nudeotide-binding folds 1 11 11 11 (NBFs). The NBFs are sufficiently similar that sequence align- NBF2 ytegGnaiLenIsFsIspGQrvgllrTGsGKs tLLsafl-r ment of highly conserved regions can be used to identify analogous residues in the two domains. To determine whether NBF1 LEPSEGKIKHSGRISFCSQFSWIMP-GTIKENI-IFGVSY-- this structural homology is paralleled in function, we compared the activation of chloride conductance by forskolin and NBF2 LlntEGeIqidGwdSitlQ-qWrkafGvIpqkvfIFsgtfrk 3-isobutyl-l-methylxanthine in Xenopus oocytes expressing CFTRs bearing mutations in NBF1 or NBF2. Mutation of a NBF1 --DEYR------YRSVIKACQLEEDISKFAEKDNIVLGEGGI conserved glycine in the putative linker domain in either NBF lIIl 11 11 produced virtually identical changes in the sensitivity of chlo- NBF2 nlDpYeqwsdqeiwkVadevgLrsvIeqFpgKldfVLvdGGc ride conductance to activating conditions, and mutation of this I, B site in both NBFs produced additive effects, suggesting that in NBF1 TLSG QRARISLARAVYKDADILYLLDSPFGYLDVLTEKEIFE the two NBFs this region plays a similar and critical role in the 11 III 1 111 I 11 activation process. In contrast, amino acid substitutions in the NBF2 vLSh hkqlmcLARsVlskAklilLLDePsahLDpvTyqiIrr Walker A and B motifs, thought to form an integral part of the FIG. 1. Amino acid sequence alignment of NBF1 (amino termi- nucleotide-binding pockets, produced strikingly different ef- nus) and NBF2 (carboxyl terminus). Amino acid sequences ofNBF1 fects in NBF1 and NBF2. Substitutions for the conserved lysine (residues 433-588) and NBF2 (residues 1219-1386) are shown in (Walker A) or aspartate (Walker B) in NBF1 resulted in a single-letter code. The bracket indicates the putative linker motif. marked decrease in sensitivity to activation, whereas the same Boxed residues indicate the Walker A motif, glycine-551 and -1349 changes in NBF2 produced an increase in sensitivity. These (located within the linker motif), and the Walker B motif. results are consistent with a model for the activation of CFTR in which both NBF1 and NBF2 are required for normal pocket to other domains of the protein (12, 13). The impor- function but in which either the nature or the exact conse- tance of this putative linker motif to CFTR function is quences of nucleotide binding differ for the two domains. evidenced by the seven CF missense mutations reported within this region of NBF1 and NBF2 (14). In a variety of cells expressing CFTR, experimental ma- Cystic fibrosis (CF) is caused by mutations in the gene neuvers that elevate cytosolic cAMP activate a Cl--selective encoding the cystic fibrosis transmembrane conductance conductance (gc) (15-24), and it is now generally accepted regulator (CFTR). Homology analysis revealed that CFTR is that CFTR functions as a Cl--selective ion channel (17, 25) a member of a superfamily of proteins referred to as traffic gated by cAMP-dependent protein kinase (PKA) and ATP ATPases (1) or ATP binding cassette (ABC) proteins (2, 3) (19, 26-29). In Xenopus oocytes expressing wild-type CFTR that includes various prokaryotic permeases as well as eu- we demonstrated (18) dose-dependent activation of Cl- con- karyotic proteins such as P-glycoprotein (4, 5). A consistent ductance by a phosphodiesterase inhibitor, 3-isobutyl-1- feature of this family is the presence of hydrophilic domains methylxanthine (IBMX), in the presence of forskolin, an having amino acid sequences characteristic of mononucle- activator of adenylate cyclase. We used the IBMX dose- otide-binding proteins. CFTR has two putative nucleotide- response to compare the conductance associated with wild- binding folds (NBFs) (6), and the amino acid sequences are type CFTR to that associated with CFTR variants bearing shown aligned in Fig. 1. Each predicted NBF contains three mutations in NBF1 (18). Certain CF-associated NBF1 mu- sequence motifs that are well conserved across the traffic tations reduced the sensitivity of CFTR to activating condi- ATPase superfamily. Two of these motifs, Walker A and B tions, and the reduction in sensitivity was correlated with the (7), are thought to form a portion of a nucleotide-binding severity of CF in patients carrying the mutations. These pocket. The conserved lysine in Walker A is predicted to findings suggested that the integrity of NBF1 was essential interact with the phosphoryl moiety of the bound nucleotide for normal activation of Cl- conductance by cAMP and that (8, 9) and the aspartic acid in Walker B to coordinate Mg2+ the reduced sensitivity to activation could be a causative of the MgATP complex (10, 11). The third motif lies between factorin disease. The IBMX dose-response appears to reflect the Walker A and B sequences and in a prokaryotic homo- the sensitivity of CFTR to increases in the activity of PKA logue, histidine permease, has been proposed to function as because reduced sensitivity of CFTR variants to IBMX is a linker that transmits signals from the nucleotide-binding Abbreviations: CF, cystic fibrosis; CFTR, cystic fibrosis transmem- The publication costs of this article were defrayed in part by page charge brane conductance regulator; NBF, nucleotide-binding fold; IBMX, payment. This article must therefore be hereby marked "advertisement" 3-isobutyl-1-methylxanthine; PKA, cAMP-dependent protein ki- in accordance with 18 U.S.C. §1734 solely to indicate this fact. nase. 9963 Downloaded by guest on September 30, 2021 9964 Physiology: Smit et al. Proc. Natl. Acad. Sci. USA 90 (1993) paralleled by reduced sensitivity to injected cAMP or cata- comparisons of activation sensitivities, CFTR variants were lytic subunit of PKA (30). expressed in the same oocyte populations. Wild-type CFTR The purpose of the present study was to examine the was tested in each population, and the results were combined functional consequences of corresponding mutations in to construct the average dose-response relationships shown NBF1 and NBF2 of CFTR by comparing the effects of these in Figs. 2-5. The greatest source of variability in determina- mutations on the sensitivity of Cl- conductance to activation tion of the normalized IBMX dose-response was the diffi- by forskolin and IBMX. The Xenopus oocyte expression culty in estimating the true value of the maximum cAMP- system offered two potential advantages: (i) dose-dependent induced Cl- conductance in oocytes expressing high levels of activation permitted a direct, quantitative comparison of the activatable CFTR. For this reason, expression levels for sensitivities of CFTR variants and (ii) mutant CFTR protein easily activated constructs (wild type, G551A, G1349A, appears less likely to be subject to intracellular processing K1250Q, and D1370N) were adjusted by reducing the amount problems in oocytes than in other cell types. For example, of injected RNA so that the maximum Cl- conductance was AF508, a mutant that exhibits a severe processing defect at similar to that attained by less sensitive constructs. The 37°C in mammalian cells (31-33), is associated with a robust IBMX dose-response relationships presented here were con- cAMP-activated Cl- conductance in oocytes (18). The results structed from experiments in which the maximum obtained with this expression system are consistent with the stimulated idea that both NBFs are required for normal activation of Cl- conductance was <100 ,LS. CFTR, but suggest that the two domains function differently in this process. RESULTS Analogous Mutations in the Putative Linker Domain. We MATERIALS AND METHODS began our functional comparison of NBF1 and NBF2 by In Vitro Mutagenesis Procedures, Plasmid Construction, and examining the effects of mutations at glycine-551 in NBF1 RNA Synthesis. To generate the mutation constructs, a 1.7-kb and the corresponding residue in NBF2, glycine-1349. These (includes NBF1 Walker A) and a 3.0-kb (includes G551, residues were chosen because both loci are associated with NBF1 Walker B, and NBF2) Sph I-Sac I CFTR cDNA naturally occurring CF mutations (14), they lie within a fragment were cloned into pSelect vector (Promega). Oligo- putative linker sequence motif, and the glycine is absolutely nucleotide-mediated site-directed mutagenesis was per- conserved throughout the traffic ATPase family (6). The formed (Altered Sites manual, Promega) using oligonucleo- effects of serine, aspartic acid, and alanine substitutions for tides corresponding to the desired mutations. The mutated glycine-551 and -1349 were compared, and the results are fragments were transferred into a full-length CFTR cDNA plotted in Fig. 2 as stimulated conductance (gcl) normalized construct (23) in pBluescript (Stratagene) and the presence of to maximum stimulated gcl versus IBMX dose. As previously the mutations was verified by sequencing. The full-length reported
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