the cystic fibrosis transmembrane conductance regulator ( cftr ) is a chloride channel in the apical membrane of epithelial cells 1 . mutations in cftr result in impaired chloride conductance across the plasma membrane and are the basis of the genetic disease cystic fibrosis 2 . almost 90% of patients contain at least one allele with a missing codon for phe508 ( f508del ) located in the first nucleotide binding domain ( nbd1 ) . cftr is regulated by phosphorylation primarily on its regulatory region ( r region ) , which is an ~200residue , disordered segment of the protein 1 . while cftr is a chloride channel , it is a member of the abc family of transporters with significant homologies in the transmembrane domains and nbds ; the r region , in contrast , is unique to cftr . the following minireview is not intended to be a comprehensive summary of the extensive and at times contradictory r region literature . instead , we focus on specific papers that highlight and enrich a mechanistic understanding of how phosphorylation affects regulation of cftr by the r region . the nterminal boundary of the r region is not precisely defined , due to lack of clarity for the cterminal boundary of the immediately nterminal nbd1 . split cftr constructs lacking the r region ( 1634 , 8381480 ) traffic to the plasma membrane and conduct chloride , suggesting that they contain a complete functional nbd1 , and thus the nterminal boundary of the r region is ~n635 4 . this is consistent with nmr data demonstrating that the cterminal residues of an nbd1 construct ending at g646 comprising helix h9 ( 637646 ) are in exchange with unfolded states and that an nbd1 construct ending at residue l636 displays stable folding 5 . however , alignment of nbd sequences from homologous abc transporters such as hisp and malk to nbd1 suggests that the domain ends at residue s642 for an nterminal r region boundary of ~k643 3 . the cterminal boundary of the r region is more defined ( about d835 ) , due to confidence in predicting the start of the immediately cterminal membranespanning domain 2 ( msd2 ) 3 . the approximately 200 residues of the r region spanning these boundaries have an amino acid composition that markedly differs from the rest of cftr , with more hydrophilic and charged residues . these features led to early recognition that the r region may be an intrinsically disordered region 6 . since then , cd spectroscopy 6 , small angle xray scattering ( saxs ) 9 and nmr spectroscopy 10 have confirmed that the isolated r region does not have a stable threedimensional fold or even stably formed secondary structure . disorder prediction software shows definitive disordered segments separated by segments that are on the discrimination limit for folded and disordered segments 9 . cd data agree on a large fraction of random coil ( 3095% ) 6 with different amounts of helical ( 546% ) and  content . different construct boundaries and experimental conditions contribute to the inherent uncertainty ; constructs with earlier nterminal boundaries have more helical content . saxs can be used to discriminate between folded , partially folded and random coil structures , and data for the r region indicate a substantial component of random coil with some partial structure 9 , incompatible with totally random coil structure 12 . while the r region is clearly disordered , it has fractional structure and clear functional roles that lead to some degree of sequence conservation among cftr proteins of different species , from fish to human 13 . disordered protein sequences lacking a threedimensional fold are not under evolutionary pressure to maintain a constrained structure ; consequently disordered regions like the r region are much more variable than folded protein domains such as the nbds . nonetheless , not only are kinase recognition sequences conserved in the r region 3 , but the distances between phosphorylation sites ( i.e. relative positions in the sequence ) are also conserved 11 . this conservation , together with the identification of a number of cysticfibrosiscausing mutations within the r region ( www.genet.sickkids.on.ca ) , highlight the importance of the r region for cftr function and regulation , including phosphorylation and potential conserved interaction segments . nmr is the most useful technique for providing residuespecific information on intrinsically disordered proteins 14 . chemical shifts are sensitive to the local electrochemical environment , with  and  carbon resonances particularly responsive to secondary structure . r region secondary structure propensity estimated on the basis of chemical shifts indicates transiently populated secondary structure elements ( fig .  1 ) 10 . the highest propensities for the helix reach 3040% , suggesting that segments 648670 , 755778 and 801818 sample helical conformation up to 3040% of the time . the flexible and dynamic features of the r region make crystallization of the r region impossible and no single structural representation can capture the r region conformational ensemble . however , xray structures of nbd1 containing some nterminal residues of the r region ( called the regulatory extension or re ) provide snapshots of the structural heterogeneity of this portion of the r region 15 and confirm the helical propensity of the nterminal segment observed by secondary structure propensity ( fig .  1 ) . the orientation of the re with respect to the nbd1 and the helical boundaries differs in the crystal structures ( 1r0x , 1xmi ) ( fig . key elements of cftr r region in ( a ) the nonphosphorylated and ( b ) the phosphorylated states . positive values correspond to helical structure sampling , while negative values refer to extended conformations . the lengths of 1433 interaction segments are set to six residues to match mode i binding to 1433 . kinase phosphorylation sites of pka ( star ) , pkc ( circle ) and ampk ( triangle ) are marked . both the secondary structure and binding experiments were carried out on nonphosphorylated ( open symbols ) and pkaphosphorylated ( solid symbols ) isolated r region ( 654838 ) . heterogeneity of cftr r region : nbd1 interactions . nterminus of the r region ( red , residues 638671 ) in different nbd1 crystal structures ( a , 1r0x ; b , 1xmi ) oriented to show the nbd dimerization interface 15 . the core nbd1 ( surface representation ) is colored as a gold gradient reflecting the proximity to atoms in the other nbd1 molecule within the nbd1:nbd1 homodimer structure ( 2pze ) . the flexible regulatory insertion ( ri ) of nbd1 that is in different conformations in the two structures these studies demonstrating disorder in the isolated r region are relevant to understanding the r region within the context of fulllength cftr . electron microscopy of cftr 17 has no discrete density for elements other than the msds and nbds homologous to sav1866 , a bacterial abc transporter , supporting the disordered nature of the r region . attempts to computationally model the r region within the context of homology models of cftr have been confounded by its disordered nature . two published models have completely distinct conformational predictions 18 for the r region . while these structures are potential conformations that the disordered r region might sample , they are unlikely to represent a stable structure populated for a significant fraction of the time importantly , the sequence characteristics and functional role as a target for posttranslational modification and as a binding partner for multiple intramolecular and intermolecular targets ( see below ) all strongly point to the disordered nature of the r region . regulatory regions in proteins are often disordered because they can easily adapt to different protein surfaces and are accessible to enzymes carrying out posttranslation modifications 20 . thus they can be sensitive to input from many partners and provide finetuning for signaling 21 . in the case of cftr r region protein kinases and phosphatases play a crucial role in the regulation of cftr primarily via controlling the level of r region phosphorylation . there is a significant body of literature on r region phosphorylation , reflecting the critical importance as well as the complexity and lack of thorough understanding of the phosphoregulatory effects , only a small subset of which can be covered here . several protein kinases , including protein kinase a ( pka ) , protein kinase c ( pkc ) and ampactivated protein kinase ( ampk ) , are known to phosphorylate the r region both in  vitro and in  vivo . of the 19 pka recognition sites predicted in the r region , 10 sites were confirmed in  vitro ( s660 , s670 , s686 , s700 , s712 , s737 , s753 , s768 , s795 , s813 ) ( fig .  1 ) 1 and six of these sites ( s660 , s700 , s712 , s737 , s795 , s813 ) were found to be phosphorylated on fulllength cftr purified from cells 2 . most of these phosphorylation sites stimulate channel activity in an additive / synergistic way ; however , s737 and s768 were reported under certain conditions to be inhibitory 24 . none of these sites individually seems to be essential for cftr regulation and mutating one , two or three of them ( s660 , s737 , s795 , s813 ) has little affect on chloride channel activity . in contrast , one study showed that mutating these four sites abolishes most of the campdependent response 6 . other studies showed that more sites had to be mutated to abolish the full response 25 , underscoring the multivalency of binding interactions , either intramolecular or intermolecular , of the r region that are affected by phosphorylation . there are six pkc consensus sequences located in the r region ( t682 , s686 , s707 , s790 , t791 , s809 ) , of which four have been phosphorylated in the isolated r region in  vitro ( s660 , s686 , s700 , s790 ) ( fig .  1 ) and one ( s686 ) was verified on the fulllength protein as well 23 . pkc phosphorylation on the r region does not stimulate the channel activity but instead enhances the effect of pka phosphorylation . interestingly , high concentrations of free calcium ( 0.5  mm ) inhibit pkc phosphorylation of the isolated r region 23 , an effect that could be on the enzymatic activity or possibly , indirectly , on the accessibility or structure of the r region itself . ampk , the cellular energy level activated kinase , phosphorylates the r region on two sites ( s737 , s768 ) ( fig . 1 ) that were previously described as inhibitory sites 24 , of which s768 has much greater impact than s737 28 . phosphorylation of these residues , which may fix cftr in a state that is insensitive to pka or pkc phosphorylation 29 , decreases the open probability of the channel by altering the channel opening but not the closing , resulting in longer interburst intervals but a similar length of each opening 30 . in contrast , another study found that pka phosphorylation could overcome the ampk inhibitory effect 28 . no single phosphatase has been demonstrated to be both necessary and sufficient to completely downregulate cftr channel activity , suggesting that cftr can be dephosphorylated by multiple phosphatases , including the protein phosphatase 2 ( pp2 ) isoforms pp2a , pp2b and pp2c and alkaline phosphatase 31 . exposing cftr to pp2a , pp2c and alkaline phosphatase reduces cftr activity more than 90% 32 ; however , pp2c deactivates the most rapidly . the various rates suggest that different phosphatases may act on functionally distinct pka sites 33 . pp2c can dephosphorylate cftr in  vitro 
 34 , can bind directly to the r region and to fulllength cftr and can deactivate cftr chloride channel 33 . pp2a directly associates with cftr segment 14511476 31 , it can dephosphorylate the r region in  vitro and a direct interaction of its regulator subunit ( rp65 ) with the r region segment 672855 has been demonstrated 36 . cftr , the three kinases pka , pkc and ampk and at least two phosphatases , pp2a and pp2c , are part of a large macromolecular complex anchored to the cytoskeleton at the apical membrane . the catalytic subunit of ampk tightly interacts with another disordered cftr segment , residues 14201457 , through a region of the kinase that normally binds the two ampk regulator subunits 32 . deleting this cftr cterminal segment has not been found to affect the biosynthesis of the channel , despite the fact that it contains two important endocytosis motifs 38 required for the interaction with ampk 32 . the b regulatory subunit of pp2a interacts with cftr residues 14511476 31 , which contain a highly conserved acidic cluster 38 . the extreme cterminal pdz motif of cftr ( 14771480 ) anchors it within the nherf ( na / h exchanger regulatory factor)ezrin a fraction of cellular pka is colocalized with cftr via ezrin , an a kinase anchoring protein ( akap ) 39 . disruption of pka  akap interactions dramatically reduced the ability of wildtype ( wt ) cftr to respond to camp modulation . the rack1 ( receptor for activated c kinase ) scaffold protein colocalizes with cftr , interacting with both the pdz1 domain of nherf1 and the activated pkc on two distinct sites 40 . moreover , rack1 is part of the pkcrack1nherf1 dimer  tubulin complex directly connected to the tubulin cytoskeleton . although cellspecific cftr targeting of pp2b has not been shown , the localization and expression of akap79 , a scaffold protein that binds pp2b , pka and pkc , support the importance of pp2b in cftr regulation 41 . in addition to ensuring that pka and pkc are in close proximity to cftr , both the nherf1ezrin  actin and the rack1nherf1 dimer investigations by sdspage , fluorescence , cd , ftir and nmr spectroscopy provide evidence for the r region becoming generally more flexible and less structured upon phosphorylation , although conflicting results have been published . data on the hydrodynamic properties include decreased r region mobility on sdspage following pka phosphorylation 23 interpreted as an apparent molecular weight that is greater than that expected from the addition of the extra phosphates and pointing to a more random and expanded structure . the r region ( 654838 ) contains almost identical numbers of positively ( 28  k / r / h ) and negatively ( 29 d / e ) charged residues , but phosphorylation increases the number of negative charges and results in charge repulsion by the increased amount of negative charge . the change in the charge balance alone , however , does not explain the absence of mobility shift upon pkc phosphorylation 23 or s768 phosphorylation or the major shift in response to s737 phosphorylation 44 . whilst computational studies 11 and the previously mentioned sdspage data 23 suggest an increase in r region size following phosphorylation , saxs data 9 point to a decrease in the radius of gyration . data on changes in secondary structure include cd experiments demonstrating an increased random coil character for the isolated r region upon pka phosphorylation 45 and ftir measurements indicating no secondary structure change in the context of fulllength cftr 46 . in contrast , marasini and coworkers showed a reduction in random coil character and highly increased helical content in isolation using cd 7 . drawing conclusions from the data is complicated by the different kinases and targeted phosphorylation sites on the r region . the finding that s737 phosphorylation has a very large effect while s768 has no impact on the secondary structure propensity of nearby segments explains the different consequences of phosphorylation of the two sites for sdspage mobility . likewise , that pkc phosphorylation of s686 and s790 has little effect on the amount of secondary structure is consistent with the comparable cd signals of the nonphosphorylated and pkcphosphorylated r region 45 . residuespecific and phosphorylationsitespecific information from nmr on the secondary structure of the highly pkaphosphorylated isolated r region indicates an overall reduction in helical structure with an increase in  content , potentially due to the repulsion between the phosphate and the partially negatively charged ctermini of helical elements ( fig .  1 ) 10 . clarification of the consequences on structural propensities and hydrodynamic behavior may rely on future sitespecific structural data from nmr on samples phosphorylated by different kinases at defined sites . the r region is much longer than required to bridge the distance between nbd1 and msd2 and the absence of a compact fold enables the r region to reach any part of cftr and into the space around the channel to contact other proteins ( fig . this allows cftr to have a large interactome , with the r region functioning as a protein interaction hub . the r region has been reported to interact with all intracellular portions of cftr including the nterminus 47 , intracellular domains 48 , nbd1 10 , nbd2 and the cterminus 49 in a phosphorylationdependent manner . the different phosphorylation states of the r region have distinct functions during channel gating and trafficking . the nonphosphorylated r region generally acts to inhibit chloride channel activity , probably by sterically blocking nbd heterodimerization 10 . nmr binding experiments showed that helical segments of the nonphosphorylated r region interact with nbd1 and that phosphorylation drastically reduces the affinity of these interactions 10 . one site of r region interaction with nbd1 , based on crystal structures of nbd1 containing the re 15 , is across the heterodimer interface ( figs  1 and 2 ) , pointing to phosphorylation being required for unhindered nbd heterodimerization and channel gating . constructs that lack the r region segment 760783 , which samples helical conformations and contains the s768 phosphorylation site , were constitutively active ( i.e. pka independent ) 50 . however , the open probability and the chloride current of split channels lacking r region segments 760783 or 708835 are only 3040% of the wt channel 50 , suggesting additional regulatory mechanisms . schematic models of ( a ) nonphosphorylated / closed channel and ( b ) pkaphosphorylated / open channel states of cftr . shown are the nterminal segment ( blue ) , membranespanning domains ( dark green ) , the intracellular domains ( yellowgreen ) , nbd1 ( yellow ) , nbd2 ( gold ) , r region ( red ) and the cterminal segment ( purple ) . disordered elements ( nterminus , r region and cterminus ) are shown as a superposition of multiple possible conformations . helix 9 of nbd1 ( residues 635643 , yellow ) and the cterminal elbow helix ( residues 842855 , green ) are also not in a fixed position to demonstrate that they can sample multiple conformations and may be considered part of the r region . the dashed line ( in b ) schematically illustrates the open channel pore , the membrane is represented as a gray bar and the r region sampled space is colored as a gradient ( red of various intensities ) . r region sampling in the two phosphorylation states is similar , although the phosphorylated r region samples space farther from the core of cftr as it is excluded from the nbd interface . nmr studies of nbd1 with the re demonstrated that interactions between the nbd1 core and the regulatory elements , the regulatory insertion ( ri ) and re ( nterminal portion of the r region ) are disrupted upon phosphorylation 51 . the release of these elements upon phosphorylation for f508del nbd1 with the re , however , is significantly smaller than for wt nbd1 , suggesting aberrant r region interactions in the mutant cftr that are  stickier and continue to block heterodimerization 51 . recent nmr studies of nbd1 demonstrate that the mutant h620q and cfft001 , a dual correctorpotentiator for cftr 5 , both enhance release of helices h8 and h9 from the nbd1 core . h8 is immediately nterminal to and h9 is arguably the beginning of the r region and both are coupled to r region segments that interact with the heterodimer interface . release of h8 and h9 by h620q or cfft001 is thus expected to potentiate cftr activity , at least in part , by reducing nbd1:r region interactions to promote nbd dimerization 52 . in addition to reducing the inhibitory effect of the nonphosphorylated r region on channel gating , phosphorylation of the r region appears to stimulate chloride conductance . the location of the r region within the protein is not critical , as moving it to the cterminus gives the same effect 50 . phosphorylation strongly increases the interaction of the r region with other parts of cftr , particularly within the portion of the protein cterminal to the r region 52 . these findings suggest that the mechanism behind the phosphorylated r region stimulatory effect probably involves association of the phosphorylated r region with other parts of cftr , possibly the n or cterminus of cftr , the intracellular domains or all of these regions . ko and coworkers reported reciprocal regulation between the solute carrier 26a ( slc26a ) family ( a3 , a6 ) and cftr 54 . slc26a3 , a chloride  bicarbonate exchanger , colocalizes with cftr at the apical membrane , markedly increasing cftr channel open probability , in a manner associated with direct interaction between the r region and the stas domain of the slc26a3 . binding of the stas and r region is enhanced by pkamediated phosphorylation of the r domain 54 , pointing to another stimulatory effect of r region phosphorylation . one possible explanation for the slc26a3mediated increase in cftr activity may be that the presence of the stas domain shifts the r region binding equilibria towards association with stas and away from interactions with cftr nbds that block productive atpdependent gating . since cftr folding and processing is inefficient , even most wt channels do not reach the plasma membrane . f508del exacerbates the folding problem , so that most f508del cftr is retained in the endoplasmic reticulum ( er ) and only a small portion of the translated protein reaches the plasma membrane 55 . although the pkaphosphorylated f508del cftr has been reported in one study to have the same open probability , its activation is at least seven times slower and its halflife at the plasma membrane is much shorter than that of wt 52 . notably , pharmacologically ertrapped wt cftr does not display the same degradation pattern as f508del 57 suggesting different intramolecular and intermolecular interactions , affecting both gating and processing . the r region interacts with other proteins during transit of the channel from the er to the plasma membrane and the posttranslational maturation of cftr is enhanced by phosphorylation 58 . cftr plasma membrane levels increase upon pka stimulation and can be blocked by the pka inhibitor h89 . these effects are associated with pkamediated phosphorylation of the r region and its interactions with 1433 proteins 58 . although 1433 prefers to bind to short phosphorylated linear motifs , it can also recognize nonphosphorylated substrates . all r region pka phosphorylation motifs seem to be involved in regulation by 1433 ( fig .  1 ) 58 . the interaction between 1433 and the phosphorylated r region might also increase heterodimerization of the nbds through competition with the r region binding equilibria , thereby increasing the folding stability of cftr and enhancing processing . most disordered proteins with a significant role in regulation contain molecular recognition elements ( mores ) , short segments that are important for recognition of binding partners 59 . mores are enriched in hydrophobic residues ( w , l , f , y ) as well as charged residues , especially arginine and aspartate 59 . the r region contains many predicted mores with different length distributions , with many related to the phosphorylation sites ( almost all pka sites contain leucines or phenylalanines and arginines ) . experimental results show that the r region has three long and three short interaction segments for nbd1 and nine short for 1433 ( fig .  1 ) the observation that there are a larger number of r region binding partners than interaction segments suggests interaction hot spots with nonexclusive binding preferences . different proteins probably compete for the same segments with the phosphorylation status of the r region and the available partners determining the actual interactions . the r region is much longer than required to bridge the distance between nbd1 and msd2 and the absence of a compact fold enables the r region to reach any part of cftr and into the space around the channel to contact other proteins ( fig . this allows cftr to have a large interactome , with the r region functioning as a protein interaction hub . the r region has been reported to interact with all intracellular portions of cftr including the nterminus 47 , intracellular domains 48 , nbd1 10 , nbd2 and the cterminus 49 in a phosphorylationdependent manner . the different phosphorylation states of the r region have distinct functions during channel gating and trafficking . the nonphosphorylated r region generally acts to inhibit chloride channel activity , probably by sterically blocking nbd heterodimerization 10 . nmr binding experiments showed that helical segments of the nonphosphorylated r region interact with nbd1 and that phosphorylation drastically reduces the affinity of these interactions 10 . one site of r region interaction with nbd1 , based on crystal structures of nbd1 containing the re 15 , is across the heterodimer interface ( figs  1 and 2 ) , pointing to phosphorylation being required for unhindered nbd heterodimerization and channel gating . constructs that lack the r region segment 760783 , which samples helical conformations and contains the s768 phosphorylation site , were constitutively active ( i.e. pka independent ) 50 . however , the open probability and the chloride current of split channels lacking r region segments 760783 or 708835 are only 3040% of the wt channel 50 , suggesting additional regulatory mechanisms . schematic models of ( a ) nonphosphorylated / closed channel and ( b ) pkaphosphorylated / open channel states of cftr . shown are the nterminal segment ( blue ) , membranespanning domains ( dark green ) , the intracellular domains ( yellowgreen ) , nbd1 ( yellow ) , nbd2 ( gold ) , r region ( red ) and the cterminal segment ( purple ) . disordered elements ( nterminus , r region and cterminus ) are shown as a superposition of multiple possible conformations . helix 9 of nbd1 ( residues 635643 , yellow ) and the cterminal elbow helix ( residues 842855 , green ) are also not in a fixed position to demonstrate that they can sample multiple conformations and may be considered part of the r region . the dashed line ( in b ) schematically illustrates the open channel pore , the membrane is represented as a gray bar and the r region sampled space is colored as a gradient ( red of various intensities ) . r region sampling in the two phosphorylation states is similar , although the phosphorylated r region samples space farther from the core of cftr as it is excluded from the nbd interface . nmr studies of nbd1 with the re demonstrated that interactions between the nbd1 core and the regulatory elements , the regulatory insertion ( ri ) and re ( nterminal portion of the r region ) are disrupted upon phosphorylation 51 . the release of these elements upon phosphorylation for f508del nbd1 with the re , however , is significantly smaller than for wt nbd1 , suggesting aberrant r region interactions in the mutant cftr that are  stickier and continue to block heterodimerization 51 . recent nmr studies of nbd1 demonstrate that the mutant h620q and cfft001 , a dual correctorpotentiator for cftr 5 , both enhance release of helices h8 and h9 from the nbd1 core . h8 is immediately nterminal to and h9 is arguably the beginning of the r region and both are coupled to r region segments that interact with the heterodimer interface . release of h8 and h9 by h620q or cfft001 is thus expected to potentiate cftr activity , at least in part , by reducing nbd1:r region interactions to promote nbd dimerization 52 . in addition to reducing the inhibitory effect of the nonphosphorylated r region on channel gating , phosphorylation of the r region appears to stimulate chloride conductance . the location of the r region within the protein is not critical , as moving it to the cterminus gives the same effect 50 . phosphorylation strongly increases the interaction of the r region with other parts of cftr , particularly within the portion of the protein cterminal to the r region 52 . these findings suggest that the mechanism behind the phosphorylated r region stimulatory effect probably involves association of the phosphorylated r region with other parts of cftr , possibly the n or cterminus of cftr , the intracellular domains or all of these regions . ko and coworkers reported reciprocal regulation between the solute carrier 26a ( slc26a ) family ( a3 , a6 ) and cftr 54 . slc26a3 , a chloride  bicarbonate exchanger , colocalizes with cftr at the apical membrane , markedly increasing cftr channel open probability , in a manner associated with direct interaction between the r region and the stas domain of the slc26a3 . binding of the stas and r region is enhanced by pkamediated phosphorylation of the r domain 54 , pointing to another stimulatory effect of r region phosphorylation . one possible explanation for the slc26a3mediated increase in cftr activity may be that the presence of the stas domain shifts the r region binding equilibria towards association with stas and away from interactions with cftr nbds that block productive atpdependent gating . since cftr folding and processing is inefficient , even most wt channels f508del exacerbates the folding problem , so that most f508del cftr is retained in the endoplasmic reticulum ( er ) and only a small portion of the translated protein reaches the plasma membrane 55 . although the pkaphosphorylated f508del cftr has been reported in one study to have the same open probability , its activation is at least seven times slower and its halflife at the plasma membrane is much shorter than that of wt 52 . notably , pharmacologically ertrapped wt cftr does not display the same degradation pattern as f508del 57 suggesting different intramolecular and intermolecular interactions , affecting both gating and processing . the r region interacts with other proteins during transit of the channel from the er to the plasma membrane and the posttranslational maturation of cftr is enhanced by phosphorylation 58 . cftr plasma membrane levels increase upon pka stimulation and can be blocked by the pka inhibitor h89 . these effects are associated with pkamediated phosphorylation of the r region and its interactions with 1433 proteins 58 . although 1433 prefers to bind to short phosphorylated linear motifs , it can also recognize nonphosphorylated substrates . all r region pka phosphorylation motifs seem to be involved in regulation by 1433 ( fig .  1 ) 58 . the interaction between 1433 and the phosphorylated r region might also increase heterodimerization of the nbds through competition with the r region binding equilibria , thereby increasing the folding stability of cftr and enhancing processing . most disordered proteins with a significant role in regulation contain molecular recognition elements ( mores ) , short segments that are important for recognition of binding partners 59 . mores are enriched in hydrophobic residues ( w , l , f , y ) as well as charged residues , especially arginine and aspartate 59 . the r region contains many predicted mores with different length distributions , with many related to the phosphorylation sites ( almost all pka sites contain leucines or phenylalanines and arginines ) . experimental results show that the r region has three long and three short interaction segments for nbd1 and nine short for 1433 ( fig .  1 ) the observation that there are a larger number of r region binding partners than interaction segments suggests interaction hot spots with nonexclusive binding preferences . different proteins probably compete for the same segments with the phosphorylation status of the r region and the available partners determining the actual interactions . an emerging picture of the r region is as a disordered hub , sampling multiple conformations rapidly and forming transient interactions with different parts of cftr and with other partners . by doing this there is likely to be overlap in the conformational space that can be sampled by non , partially or highly phosphorylated r region . figure  3 demonstrates the conformational heterogeneity of the r region in a model of nonphosphorylated cftr , representing a closed channel state . in this state the nbds are probably separated ; therefore the nterminus of the r region easily moves between the nbds and samples similar conformations to those found in the nbd1 xray structures ( fig .  2 ) . this segment can also interact with other parts of the nbds or other partners farther from the dimerization interface . the rest of the r region also samples a significant volume of space with the ability to make contact points with the nbds . figure  3 illustrates a model of phosphorylated cftr , representing an open channel state , with the nbds tightly associated . in a highly pkaphosphorylated state , the r region is expected to sample a similar volume of space as in nonphosphorylated cftr , although it is probably excluded from the nbd dimerization interface and therefore can extend farther away from the core of cftr ( fig . while this does not agree with hydrodynamic data from saxs for the isolated r region , the increased  character observed by nmr , the reduction of its interactions with the nbds and increased binding for the slc26a3/6 stas domains and 1433 suggest that the phosphorylated r region may sample more extended conformations within the context of fulllength cftr . that the r region can sample similar space in either phosphorylation state and that in  vivo cftr has some degree of basal phosphorylation suggest that most of the binding partners interact with a variety of phosphorylation states of the r region , but with modulated affinities . one example is the interaction with 1433 , which interacts more weakly with the nonphosphorylated state and more strongly following phosphorylation 58 . thus , r region phosphorylation is not a sharp switch but rather functions to precisely tune competing interactions . many cftr interacting proteins at the plasma membrane are located in the same subcellular compartment as cftr . moreover , most of these are components of the same macromolecular complex anchored to the cytoskeleton . activators , inhibitors , modifiers and other members of the complex enable cftr to be readily responsive to different environmental conditions . the proposed view of the r region as a dynamic hub suggests that it plays a dominant role , scanning the surrounding protein surfaces and making transient interactions with affinities and lifetimes dependent on its phosphorylation state . the plasticity of this disordered region of cftr is crucial for the complex regulatory control of cftr channel activity and its other biological functions in maintaining ion balance at the cell membrane .