Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Cl− Channel by Its R Domain (2001)

cystic fibrosis transmembrane conductance regulator ATP-binding cassette nucleotide binding domain membrane-spanning domain protein kinase A amino acid(s) kinase-inducible domain cAMP-responsive element-binding protein CREB-binding protein Since the sequence of the CFTR1 Cl− channel was discovered, the function of its R domain has been puzzling. CFTR is a member of the ATP-binding cassette (ABC) transporter family, and it contains the features characteristic of this family: two nucleotide binding domains (NBDs) and two membrane-spanning domains (MSDs) (1Riordan J.R. Rommens J.M. Kerem B.S. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Drumm M.L. Iannuzzi M.C. Collins F.S. Tsui L.C. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5940) Google Scholar). Yet in addition, CFTR also contains the R domain, a unique sequence not found in other ABC transporters or any other proteins. The R domain serves as the major physiologic regulator of the CFTR Cl− channel (2Sheppard D.N. Welsh M.J. Physiol. Rev. 1999; 79: S23-S45Crossref PubMed Scopus (803) Google Scholar, 3Gadsby D.C. Nairn A.C. Physiol. Rev. 1999; 79: S77-S107Crossref PubMed Scopus (369) Google Scholar). Upon elevation of cAMP levels, cAMP-dependent protein kinase (PKA) phosphorylates the R domain allowing ATP to open and close the channel. Yet how phosphorylation activates the channel is not well understood. Some models propose that the R domain prevents the channel from opening and that phosphorylation relieves this inhibition. Other models suggest that phosphorylation of the R domain stimulates activity. Here we briefly review current knowledge that may help explain the function of this interesting domain. The R domain was originally defined as those residues encoded by exon 13 (aa 590–830) (Fig. 1) (1Riordan J.R. Rommens J.M. Kerem B.S. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Drumm M.L. Iannuzzi M.C. Collins F.S. Tsui L.C. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5940) Google Scholar). Although the precise boundaries of the R domain remain uncertain, recent studies suggest that the N-terminal portion of exon 13 is actually part of NBD1, whereas the C-terminal portion constitutes the structural and functional "R domain." Supporting this conclusion, the N-terminal portion of exon 13 has sequence similarity with NBDs of other ABC transporters (4Rich D.P. Gregory R.J. Anderson M.P. Manavalan P. Smith A.E. Welsh M.J. Science. 1991; 253: 205-207Crossref PubMed Scopus (194) Google Scholar, 5Dulhanty A.M. Riordan J.R. FEBS Lett. 1994; 343: 109-114Crossref PubMed Scopus (30) Google Scholar). More recently, crystal structures were solved for the NBDs of the ABC transporters HisP, MalK, and Rad50 (6Hung L.W. Wang I.X. Nikaido K. Liu P.Q. Ames G.F.L. Kim S.H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (618) Google Scholar, 7Hopfner K.P. Karcher A. Shin D.S. Craig L. Arthur L.M. Carney J.P. Tainer J.A. Cell. 2000; 101: 789-800Abstract Full Text Full Text PDF PubMed Scopus (810) Google Scholar, 8Diederichs K. Diez J. Greller G. Muller C. Breed J. Schnell C. Vonrhein C. Boos W. Welte W. EMBO J. 2000; 19: 5951-5961Crossref PubMed Scopus (276) Google Scholar). In CFTR, residues analogous to those NBDs would extend to approximately aa 642. Experimental evidence for the boundaries came with the demonstration that deletion of aa 708–835, but not deletions extending further in the N-terminal direction, produced a channel that was processed correctly, opened in the presence of ATP, and had conductive properties like those of wild-type CFTR (4Rich D.P. Gregory R.J. Anderson M.P. Manavalan P. Smith A.E. Welsh M.J. Science. 1991; 253: 205-207Crossref PubMed Scopus (194) Google Scholar, 9Rich D.P. Gregory R.J. Cheng S.H. Smith A.E. Welsh M.J. Receptors Channels. 1993; 1: 221-232PubMed Google Scholar). In addition, severing CFTR after residues 633 or 835 and coexpressing the two halves in Xenopus oocytes produced functional channels, whereas severing the channel between exons 12 and 13 abolished function (10Chan K.W. Csanady L. Seto-Young D. Nairn A.C. Gadsby D.C. J. Gen. Physiol. 2000; 116: 163-180Crossref PubMed Scopus (70) Google Scholar,11Csanady L. Chan K.W. Seto-Young D. Kopsco D.C. Nairn A.C. Gadsby D.C. J. Gen. Physiol. 2000; 116: 477-500Crossref PubMed Scopus (99) Google Scholar). All these data suggest that the N-terminal R domain boundary begins between residues 634 and 708 and that the C-terminal boundary ends in the region of residue 835. The most striking feature of the R domain is the presence of multiple consensus PKA phosphorylation sites that are highly conserved across species (Fig. 1). There is little other sequence similarity in the R domain, unlike the substantial sequence conservation throughout the rest of CFTR. We are not aware of any other conserved protein motifs in the R domain, although there is a relatively high percentage (28%) of charged residues. Sixteen of the 24 basic residues are in PKA consensus sites. Several of the acidic residues are clustered within 817–838 (12Xie J. Zhao J. Davis P.B. Ma J. Biophys. J. 2000; 78: 1293-1305Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). PKA is the primary kinase that phosphorylates CFTR, although protein kinase C, cGMP-dependent protein kinase, and tyrosine phosphorylation can also stimulate channel activity (2Sheppard D.N. Welsh M.J. Physiol. Rev. 1999; 79: S23-S45Crossref PubMed Scopus (803) Google Scholar, 3Gadsby D.C. Nairn A.C. Physiol. Rev. 1999; 79: S77-S107Crossref PubMed Scopus (369) Google Scholar).In vitro, PKA phosphorylates Ser-660, Ser-700, Ser-712, Ser-737, Ser-753, Ser-768, Ser-795, and Ser-813 (13Cheng S.H. Rich D.P. Marshall J. Gregory R.J. Welsh M.J. Smith A.E. Cell. 1991; 66: 1027-1036Abstract Full Text PDF PubMed Scopus (532) Google Scholar, 14Picciotto M.R. Cohn J.A. Bertuzzi G. Greengard P. Nairn A.C. J. Biol. Chem. 1992; 267: 12742-12752Abstract Full Text PDF PubMed Google Scholar). PKA also phosphorylates these residues plus Ser-686 in isolated R domain proteins or peptides (14Picciotto M.R. Cohn J.A. Bertuzzi G. Greengard P. Nairn A.C. J. Biol. Chem. 1992; 267: 12742-12752Abstract Full Text PDF PubMed Google Scholar, 15Seibert F.S. Tabcharani J.A. Chang X.B. Dulhanty A.M. Mathews C. Hanrahan J.W. Riordan J.R. J. Biol. Chem. 1995; 270: 2158-2162Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 16Neville D.C.A. Rozanas C.R. Tulk B.M. Townsend R.R. Verkman A.S. Biochemistry. 1998; 37: 2401-2409Crossref PubMed Scopus (38) Google Scholar). In contrast, when CFTR is phosphorylated in cells by addition of cAMP agonists, detectable phosphorylation is limited to Ser-660, Ser-700, Ser-737, Ser-795, and Ser-813 (13Cheng S.H. Rich D.P. Marshall J. Gregory R.J. Welsh M.J. Smith A.E. Cell. 1991; 66: 1027-1036Abstract Full Text PDF PubMed Scopus (532) Google Scholar, 14Picciotto M.R. Cohn J.A. Bertuzzi G. Greengard P. Nairn A.C. J. Biol. Chem. 1992; 267: 12742-12752Abstract Full Text PDF PubMed Google Scholar,17Cohn J.A. Nairn A.C. Marino C.R. Melhus O. Kole J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2340-2344Crossref PubMed Scopus (71) Google Scholar). The functional consequences of phosphorylation have been evaluated by studying CFTR mutated at one or many of the phosphorylatable serines (13Cheng S.H. Rich D.P. Marshall J. Gregory R.J. Welsh M.J. Smith A.E. Cell. 1991; 66: 1027-1036Abstract Full Text PDF PubMed Scopus (532) Google Scholar, 15Seibert F.S. Tabcharani J.A. Chang X.B. Dulhanty A.M. Mathews C. Hanrahan J.W. Riordan J.R. J. Biol. Chem. 1995; 270: 2158-2162Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 18Rich D.P. Berger H.A. Cheng S.H. Travis S.M. Saxena M. Smith A.E. Welsh M.J. J. Biol. Chem. 1993; 268: 20259-20267Abstract Full Text PDF PubMed Google Scholar, 19Chang X.B. Tabcharani J.A. Hou Y.X. Jensen T.J. Kartner N. Alon N. Hanrahan J.W. Riordan J.R. J. Biol. Chem. 1993; 268: 11304-11311Abstract Full Text PDF PubMed Google Scholar, 20Winter M.C. Welsh M.J. Nature. 1997; 389: 294-296Crossref PubMed Scopus (124) Google Scholar, 21Wilkinson D.J. Strong T.V. Mansoura M.K. Wood D.L. Smith S.S. Collins F.S. Dawson D.C. Am. J. Physiol. 1997; 273: L127-L133PubMed Google Scholar, 22Mathews C.J. Tabcharani J.A. Chang X.B. Jensen T.J. Riordan J.R. Hanrahan J.W. J. Physiol. 1998; 508: 365-377Crossref PubMed Scopus (63) Google Scholar, 23Baldursson O. Berger H.A. Welsh M.J. Am. J. Physiol. 2000; 279: L835-L841Crossref PubMed Google Scholar). Serine mutations reduced but did not abolish phosphorylation-stimulated activity, and no single serine was required for stimulation. Even when all the consensus phosphoserines were mutated, a small amount of PKA-mediated Cl− current remained, suggesting a contribution from sites that are not normally phosphorylated in vivo such as Ser-753 (15Seibert F.S. Tabcharani J.A. Chang X.B. Dulhanty A.M. Mathews C. Hanrahan J.W. Riordan J.R. J. Biol. Chem. 1995; 270: 2158-2162Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 19Chang X.B. Tabcharani J.A. Hou Y.X. Jensen T.J. Kartner N. Alon N. Hanrahan J.W. Riordan J.R. J. Biol. Chem. 1993; 268: 11304-11311Abstract Full Text PDF PubMed Google Scholar). Nonetheless, the majority of PKA-dependent stimulation appears to result from phosphorylation of Ser-660, Ser-700, Ser-737, Ser-795, and Ser-813. Although no one serine is essential, individual serines seem to stimulate channel activity to different degrees. For example, mutation of either Ser-660 or Ser-813 reduced the open state probability (Po) of CFTR studied under several conditions (20Winter M.C. Welsh M.J. Nature. 1997; 389: 294-296Crossref PubMed Scopus (124) Google Scholar,21Wilkinson D.J. Strong T.V. Mansoura M.K. Wood D.L. Smith S.S. Collins F.S. Dawson D.C. Am. J. Physiol. 1997; 273: L127-L133PubMed Google Scholar, 23Baldursson O. Berger H.A. Welsh M.J. Am. J. Physiol. 2000; 279: L835-L841Crossref PubMed Google Scholar). Thus, these two residues play key stimulatory roles. Phosphorylation of Ser-795 also stimulated, although to a lesser extent (21Wilkinson D.J. Strong T.V. Mansoura M.K. Wood D.L. Smith S.S. Collins F.S. Dawson D.C. Am. J. Physiol. 1997; 273: L127-L133PubMed Google Scholar, 23Baldursson O. Berger H.A. Welsh M.J. Am. J. Physiol. 2000; 279: L835-L841Crossref PubMed Google Scholar). Interestingly, the consequences of mutating Ser-737 differed depending upon how it was studied. In excised, cell-free patches, the S737A mutant reduced Po (20Winter M.C. Welsh M.J. Nature. 1997; 389: 294-296Crossref PubMed Scopus (124) Google Scholar), but inXenopus oocytes and epithelia, the S737A mutant increased cAMP-stimulated current (21Wilkinson D.J. Strong T.V. Mansoura M.K. Wood D.L. Smith S.S. Collins F.S. Dawson D.C. Am. J. Physiol. 1997; 273: L127-L133PubMed Google Scholar, 23Baldursson O. Berger H.A. Welsh M.J. Am. J. Physiol. 2000; 279: L835-L841Crossref PubMed Google Scholar). We speculate that these differences may be because of loss of kinases, phosphatases, or associated molecules after membrane excision. Various phosphoserines also show functional interactions. For example, mutating either Ser-660 or Ser-813 alone reduced phosphorylation-stimulated activity. However, when either Ser-660 or Ser-813 was present in a variant in which the other phosphorylation sites were mutated, current did not increase; both Ser-660 and Ser-813 were essential for current to reach wild-type levels (23Baldursson O. Berger H.A. Welsh M.J. Am. J. Physiol. 2000; 279: L835-L841Crossref PubMed Google Scholar). Likewise, Ser-737 was unable to stimulate or inhibit unless other serines were also present (21Wilkinson D.J. Strong T.V. Mansoura M.K. Wood D.L. Smith S.S. Collins F.S. Dawson D.C. Am. J. Physiol. 1997; 273: L127-L133PubMed Google Scholar, 23Baldursson O. Berger H.A. Welsh M.J. Am. J. Physiol. 2000; 279: L835-L841Crossref PubMed Google Scholar). These data suggest either concomitant or sequential phosphorylation events may be required. Additional clues to R domain function came from studies of R domain deletions. Deleting aa 708–835 (ΔR) or 768–830 (Fig.2) generated channels that were open in the presence of ATP but did not require phosphorylation,i.e. they were constitutively active (4Rich D.P. Gregory R.J. Anderson M.P. Manavalan P. Smith A.E. Welsh M.J. Science. 1991; 253: 205-207Crossref PubMed Scopus (194) Google Scholar, 24Ma J. Zhao J. Drumm M.L. Xie J. Davis P.B. J. Biol. Chem. 1997; 272: 28133-28141Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 25Vankeerberghen A. Lin W. Jaspers M. Cuppens H. Nilius B. Cassiman J.J. Biochemistry. 1999; 38: 14988-14998Crossref PubMed Scopus (16) Google Scholar). These data suggested that the unphosphorylated R domain inhibits activity and that phosphorylation or deletion eliminates the inhibition. This led to the proposal that the R domain might be an inhibitory particle, much like the N terminus of the Shaker K+ channel (26Hoshi T. Zagotta W.N. Aldrich R.W. Science. 1990; 250: 533-538Crossref PubMed Scopus (1277) Google Scholar). However, that hypothesis is difficult to reconcile with data showing that the mere presence of an R domain is insufficient to inhibit constitutive activity. For example, coexpressing two halves of CFTR (aa 1–835 and aa 837–1480) that retain the R domain generated a constitutively active channel (Fig. 2) (11Csanady L. Chan K.W. Seto-Young D. Kopsco D.C. Nairn A.C. Gadsby D.C. J. Gen. Physiol. 2000; 116: 477-500Crossref PubMed Scopus (99) Google Scholar, 27King S.A. Sorscher E.J. Biochemistry. 2000; 39: 9868-9875Crossref PubMed Scopus (13) Google Scholar). Likewise, translocating aa 709–835 to the C terminus of CFTR-ΔR produced constitutively active channels (28Baldursson O. Ostedgaard L.S. Rokhlina T. Cotten J.F. Welsh M.J. J. Biol. Chem. 2001; 276: 1904-1910Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Moreover, adding isolated unphosphorylated R domain proteins encompassing aa 645–834, 590–858, or 708–831 did not reduce constitutive activity of CFTR-ΔR (20Winter M.C. Welsh M.J. Nature. 1997; 389: 294-296Crossref PubMed Scopus (124) Google Scholar, 24Ma J. Zhao J. Drumm M.L. Xie J. Davis P.B. J. Biol. Chem. 1997; 272: 28133-28141Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 29Ostedgaard L.S. Baldursson O. Vermeer D.W. Welsh M.J. Robertson A.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5657-5662Crossref PubMed Scopus (94) Google Scholar). To identify R domain sequences that might prevent constitutive activity, other deletions were studied. A smaller R domain deletion (aa 760–835) also induced constitutive activity, whereas deletions of residues 708–759, 784–835, and 780–830 did not (Fig. 2) (25Vankeerberghen A. Lin W. Jaspers M. Cuppens H. Nilius B. Cassiman J.J. Biochemistry. 1999; 38: 14988-14998Crossref PubMed Scopus (16) Google Scholar, 28Baldursson O. Ostedgaard L.S. Rokhlina T. Cotten J.F. Welsh M.J. J. Biol. Chem. 2001; 276: 1904-1910Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). This focused attention on residues 760–783; deletion of these residues generated a constitutively active channel (28Baldursson O. Ostedgaard L.S. Rokhlina T. Cotten J.F. Welsh M.J. J. Biol. Chem. 2001; 276: 1904-1910Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). There are no obvious sequence motifs between aa 760 and 783, although the sequence765RRQSVL(N/D)LMT774 is conserved across species. Ser-768 is located in this region, but it is not phosphorylated in vivo (13Cheng S.H. Rich D.P. Marshall J. Gregory R.J. Welsh M.J. Smith A.E. Cell. 1991; 66: 1027-1036Abstract Full Text PDF PubMed Scopus (532) Google Scholar, 14Picciotto M.R. Cohn J.A. Bertuzzi G. Greengard P. Nairn A.C. J. Biol. Chem. 1992; 267: 12742-12752Abstract Full Text PDF PubMed Google Scholar), and its mutation did not induce constitutive activity (21Wilkinson D.J. Strong T.V. Mansoura M.K. Wood D.L. Smith S.S. Collins F.S. Dawson D.C. Am. J. Physiol. 1997; 273: L127-L133PubMed Google Scholar, 28Baldursson O. Ostedgaard L.S. Rokhlina T. Cotten J.F. Welsh M.J. J. Biol. Chem. 2001; 276: 1904-1910Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Interestingly, adding residues 760–783 as isolated peptides or translocating them to the C terminus failed to prevent constitutive activity (Fig. 2). Thus, the R domain probably has an inhibitory function that requires a small portion in the middle of the domain. However, those residues alone are not sufficient to prevent constitutive activity, and to have an effect it appears that they must be retained in their normal location between NBD1 and MSD2. Direct evidence that the phosphorylated R domain is stimulatory came from studies that added isolated R domain proteins to CFTR-ΔR channels. Phosphorylated, but not unphosphorylated, proteins consisting of residues 645–834, 590–858, and 708–831 each stimulated Cl− current (Fig. 2) (20Winter M.C. Welsh M.J. Nature. 1997; 389: 294-296Crossref PubMed Scopus (124) Google Scholar, 24Ma J. Zhao J. Drumm M.L. Xie J. Davis P.B. J. Biol. Chem. 1997; 272: 28133-28141Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 29Ostedgaard L.S. Baldursson O. Vermeer D.W. Welsh M.J. Robertson A.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5657-5662Crossref PubMed Scopus (94) Google Scholar). In addition, translocating aa 709–835, 709–759, or 760–835 to the C terminus restored PKA-stimulated activity to CFTR-ΔR (28Baldursson O. Ostedgaard L.S. Rokhlina T. Cotten J.F. Welsh M.J. J. Biol. Chem. 2001; 276: 1904-1910Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). One study suggesting that the R domain is not stimulatory found that channels generated by coexpressing aa 3–633 and 837–1480 opened as often as wild type (Fig. 2) (11Csanady L. Chan K.W. Seto-Young D. Kopsco D.C. Nairn A.C. Gadsby D.C. J. Gen. Physiol. 2000; 116: 477-500Crossref PubMed Scopus (99) Google Scholar). However, the Po of those severed channels was only half that of wild-type channels. Another study found that deleting the negatively charged region at aa 817–838 generated a constitutively active channel whose activity was not increased by PKA (Fig. 2) (12Xie J. Zhao J. Davis P.B. Ma J. Biophys. J. 2000; 78: 1293-1305Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). This finding suggested that this relatively conserved region was required for stimulation. These data clearly indicate that the R domain can stimulate activity, although the mechanism is unknown. Interestingly, stimulation can come from multiple R domain regions, more than one phosphoserine can stimulate, and no one specific phosphoserine or unique sequence is required. The data also suggest that stimulatory regions differ from those preventing constitutive activity. Phosphorylation raises Po by increasing the rate at which the channel opens (20Winter M.C. Welsh M.J. Nature. 1997; 389: 294-296Crossref PubMed Scopus (124) Google Scholar, 22Mathews C.J. Tabcharani J.A. Chang X.B. Jensen T.J. Riordan J.R. Hanrahan J.W. J. Physiol. 1998; 508: 365-377Crossref PubMed Scopus (63) Google Scholar, 24Ma J. Zhao J. Drumm M.L. Xie J. Davis P.B. J. Biol. Chem. 1997; 272: 28133-28141Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Phosphorylation also increases the apparent affinity of the NBDs for ATP; this effect differs for different phosphoserines (20Winter M.C. Welsh M.J. Nature. 1997; 389: 294-296Crossref PubMed Scopus (124) Google Scholar, 22Mathews C.J. Tabcharani J.A. Chang X.B. Jensen T.J. Riordan J.R. Hanrahan J.W. J. Physiol. 1998; 508: 365-377Crossref PubMed Scopus (63) Google Scholar). In general the more phosphorylation, the greater the activity. This is likely to be of physiologic importance, as graded phosphorylation could generate graded apical membrane Cl− channel activity allowing precise control of transepithelial Cl− transport. To better understand the functional and structural features of this unique domain, we expressed and purified an R domain protein comprising aa 708–831 (named R8) (29Ostedgaard L.S. Baldursson O. Vermeer D.W. Welsh M.J. Robertson A.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5657-5662Crossref PubMed Scopus (94) Google Scholar). Analytical ultracentrifugation showed that soluble R8 was a monomer, and limited proteolysis indicated that most of R8 was accessible to proteases. Phosphorylation did not change these properties, suggesting that it did not induce global conformational changes. Importantly, phosphorylated R8 stimulated channel activity, suggesting that if the R domain has a specific structure, it is retained in this protein. Circular dichroism (CD) suggested that R8 had little well ordered secondary structure; helical content was about 5% with the remaining protein being random coil (29Ostedgaard L.S. Baldursson O. Vermeer D.W. Welsh M.J. Robertson A.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5657-5662Crossref PubMed Scopus (94) Google Scholar). The results were the same irrespective of whether spectra were obtained in high pH, low salt, buffers used for patch-clamp studies, trimethylamine oxide (which promotes protein folding), or R8 incorporated in phospholipid micelles. Phosphorylated and nonphosphorylated R8 had similar CD spectra, suggesting that phosphorylation did not induce large structural changes. CD spectra of larger R domain proteins including all or parts of NBD1 revealed significant secondary structure; aa 595–831 had 10% α-helix and 30% β-sheet (30Dulhanty A.M. Riordan J.R. Biochemistry. 1994; 33: 4072-4079Crossref PubMed Scopus (94) Google Scholar), whereas aa 404–830 had 19% α-helix and 43% β-sheet (16Neville D.C.A. Rozanas C.R. Tulk B.M. Townsend R.R. Verkman A.S. Biochemistry. 1998; 37: 2401-2409Crossref PubMed Scopus (38) Google Scholar). The secondary structure in those proteins may arise from the NBD1 portion and the random coil from the R domain. Although other ABC transporters do not contain a region homologous to the R domain, P-glycoprotein contains a central region connecting NBD1 and MSD2 that includes two PKA motifs. Deleting this linker region (aa 653–686) disrupted drug transport and ATP hydrolysis (31Hrycyna C.A. Airan L.E. Germann U.A. Ambudkar S.V. Pastan I. Gottesman M.M. Biochemistry. 1998; 37: 13660-13673Crossref PubMed Scopus (94) Google Scholar). Replacing the linker with an unrelated sequence predicted to form a flexible structure restored both drug transport and ATP hydrolysis. Interestingly, when linker residues 644–689 from P-glycoprotein replaced aa 780–830 in CFTR, stimulated channel activity was restored to wild-type levels (25Vankeerberghen A. Lin W. Jaspers M. Cuppens H. Nilius B. Cassiman J.J. Biochemistry. 1999; 38: 14988-14998Crossref PubMed Scopus (16) Google Scholar). These observations suggest that a defined amino acid sequence is not critical, but PKA consensus motifs, a certain degree of flexibility, and possibly an optimal length are key in determining channel activity. Several other observations support the conclusion that the functional R domain is predominantly random coil. First, the entire R domain was neither required to stimulate activity nor to prevent constitutive activity; portions were sufficient (Fig. 2). the R domain had a well ordered structure, such would likely be the R domain sequence is not well conserved across species (Fig. 1). highly domains show substantial sequence are the Moreover, many of the conserved residues in such proteins have within the protein they help structure J.F. Science. 1990; PubMed Scopus Google Scholar). In contrast, the conserved R domain sequences are consensus PKA phosphorylation motifs that are to be they functional than structural roles. of the more than mutations and in the CFTR only between aa 708 and two are at conserved PKA motifs (Fig. 1). that not all of these sequence are actually to cystic fibrosis because is these for the that have been their not differ from wild-type A. L. Jaspers M. Cassiman J.J. Nilius B. Cuppens H. 1998; PubMed Scopus Google Scholar), with the that an amino acid change in random coil would be to Moreover, these show relatively small functional differences A. L. Jaspers M. Cassiman J.J. Nilius B. Cuppens H. 1998; PubMed Scopus Google Scholar). the R domain is predominantly random coil and if phosphorylation not structure, how the R domain the There are several Phosphorylation might induce a conformational change but only in small of by phosphorylation may be sufficient to activity; with of serines with stimulates activity D.P. Berger H.A. Cheng S.H. Travis S.M. Saxena M. Smith A.E. Welsh M.J. J. Biol. Chem. 1993; 268: 20259-20267Abstract Full Text PDF PubMed Google Scholar). Phosphorylation may not change but may be required the R domain can with other sites within CFTR. The R domain may a more ordered structure only upon with the rest of CFTR. The two are of the of the kinase-inducible domain of the cAMP-responsive element-binding protein with CREB-binding protein Several structural indicate that is and serine phosphorylation not structure J.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, I. D. M.R. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, I. FEBS Lett. 1998; PubMed Scopus Google Scholar, D. K. T. R. C. M.R. Cell. Biol. PubMed Scopus Google Scholar, D. M. T. A. I. A. M. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar). However, as phosphorylation the R domain to the phosphorylation to and This binding a conformational change in which is limited to the phosphoserine and a amino a phosphorylated random coil R domain stimulate models (29Ostedgaard L.S. Baldursson O. Vermeer D.W. Welsh M.J. Robertson A.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5657-5662Crossref PubMed Scopus (94) Google Scholar). The phosphorylated R domain might with a single or multiple sites and on the rest of CFTR. may be specific or The of phosphoserines and sites is and for In this we the of an but phosphorylated R domain for stimulation. However, the not show how an unphosphorylated R domain prevents constitutive activity. current of it difficult to both stimulatory and inhibitory in the same A the unphosphorylated R domain as a random coil. phosphorylation, it not stimulate activity. a phosphorylated R domain. the R domain is any of several different phosphoserines could with the single to stimulate activity. could explain the of multiple different phosphoserines to stimulate, possibly with different on activity. multiple phosphoserines with unique sites in CFTR. This could for increasing activity with increasing if there were or there would be or sites. This could also for different generated by phosphorylation of different multiple phosphoserines with multiple binding sites. Here we show only suggesting the of at one at each However, more sites may as by the that regions within the R domain and the N terminus J. M. Science. 1999; PubMed Scopus Google Scholar). could for the apparent between phosphorylation sites and stimulation of activity as by the In such a a relatively R domain would multiple different phosphoserines to stimulate, no one phosphoserine would be different phosphoserines could have different and the more serines the greater the activity. There is increasing that many protein domains and proteins are K.W. M. Nature. 1997; PubMed Scopus Google Scholar, J. Biol. 1999; PubMed Scopus Google Scholar). the kinase R.W. L. L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), the protein of Biol. 1997; PubMed Scopus Google Scholar), and the protein C.J. C. I. J. D.L. Smith Smith J. Biol. 1997; PubMed Scopus Google Scholar). Interestingly, a sequence in the domain of the Shaker K+ also in a Aldrich R.W. J. Gen. Physiol. 1993; PubMed Scopus Google Scholar). The of proteins for or are also more apparent K.W. M. Nature. 1997; PubMed Scopus Google Scholar, J. Biol. 1999; PubMed Scopus Google Scholar). In CFTR, an R domain predominantly of random coil would retain the that with multiple regions within the rest of CFTR and and to phosphorylation of different study on the R domain, a of is of PKA motifs conserved within random are certain regions within the R domain but not sufficient for and those residues be within the and retain is the mechanism by which the R domain stimulates and inhibits the We for and for