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Cystic Fibrosis Transmembrane Conductance Regulator Cl−Channels with R Domain Deletions and Translocations Show Phosphorylation-dependent and -independent Activity

Olafur Baldursson, Lynda S. Ostedgaard, Tatiana Rokhlina, Joseph F. Cotten, Michael J. Welsh
2000 Journal of Biological Chemistry  
Phosphorylation of the R domain regulates cystic fibrosis transmembrane conductance regulator Cl ؊ channel activity. Earlier studies suggested that the R domain controls activity via more than one mechanism; a phosphorylated R domain may stimulate activity, and an unphosphorylated R domain may prevent constitutive activity, i.e. opening with ATP alone. However, the mechanisms responsible for these two regulatory properties are not understood. In this study we asked whether the two effects are
more » ... e two effects are dependent on its position in the protein and whether smaller regions from the R domain mediate the effects. We found that several portions of the R domain conferred phosphorylation-stimulated activity. This was true whether the R domain sequences were present in their normal location or were translocated to the C terminus. We also found that some parts of the R domain could be deleted without inducing constitutive activity. However, when residues 760 -783 were deleted, channels opened without phosphorylation. Translocation of the R domain to the C terminus did not prevent constitutive activity. These results suggest that different parts of the phosphorylated R domain can stimulate activity and that their location within the protein is not critical. In contrast, prevention of constitutive activity required a short specific sequence that could not be moved to the C terminus. These results are consistent with a recent model of an R domain composed primarily of random coil in which more than one phosphorylation site is capable of stimulating channel activity, and net activity reflects interactions between multiple sites in the R domain and the rest of the channel. The cystic fibrosis transmembrane conductance regulator (CFTR) 1 Cl Ϫ channel contains three cytosolic domains as follows: a regulatory R domain and two nucleotide-binding domains (NBDs). ATP binding and hydrolysis by the NBDs gate the channel, and phosphorylation of the R domain regulates activity (for reviews see Refs. 1 and 2). Although the boundaries of the R domain are not known precisely, several lines of evidence suggest that this domain extends from approximately residues 700 to 830 (3-6). Within this region, we found that the cAMP-dependent protein kinase (PKA) phosphorylates three serines (residues 737, 795, and 813) in intact cells to regulate channel activity (7). Picciotto and colleagues (8) found that these residues plus residue 700 were phosphorylated in vivo. Outside this region, Ser-660 is phosphorylated by PKA and contributes to channel regulation (7, 8). Studies of CFTR variants in which the phosphoserines have been mutated to alanine show that phosphorylation of multiple R domain serines contributes to regulation, although no one serine is required for activity (for reviews see Refs. 1 and 2). Further insight into R domain structure and function has come from studies in which the R domain has been deleted. Such studies have suggested that the R domain may have two functions as follows: the unphosphorylated R domain may prevent constitutive activity (i.e. opening in the presence of ATP alone), and the phosphorylated R domain may stimulate activity. Speculation that the unphosphorylated R domain prevents constitutive activity has been based primarily on studies of CFTR in which residues 708 -835 have been deleted. Expression of this variant generated a channel that was constitutively open; only ATP was required for activity, and PKA-dependent phosphorylation was not required (9, 10). Larger deletions that extended the N terminus of residue 708 failed to generate channels, likely because of a severe disruption of structure (3). There have been only two reports of a smaller deletion, and although the portion deleted was similar in the two studies, the results were different (11, 12) . The R domain can also stimulate channel activity. Addition of a phosphorylated recombinant R domain protein stimulated activity of CFTR in which residues 708 -835 were deleted. Phosphoproteins consisting of residues 645-834, 590 -858, and 708 -831 each stimulated Cl Ϫ current (6, 10, 13). Thus it appears that the R domain may both prevent constitutive activity and, when phosphorylated, stimulate activity. However, it is not clear whether different portions of the R domain mediate these functions. To investigate these functions, we took two approaches; we deleted portions of the R domain, and we translocated portions of the R domain to the C terminus of the channel. We then asked how constitutive and stimulated activity were affected. EXPERIMENTAL PROCEDURES Chemicals and Solutions-The catalytic subunit of PKA was obtained from Promega (Madison, WI) and ATP from Sigma. Protein
doi:10.1074/jbc.m006934200 pmid:11038358 fatcat:ugvv5p4nwrbmfh2h2bgy5q5ole