Dynamic Regulation of Cystic Fibrosis Transmembrane Conductance Regulator by Competitive Interactions of Molecular Adaptors

Disorganized ion transport caused by hypo- or hyperfunctioning of the cystic fibrosis transmembrane conductance regulator (CFTR) can be detrimental and may result in life-threatening diseases such as cystic fibrosis or secretory diarrhea. Thus, CFTR is controlled by elaborate positive and negative regulations for an efficient homeostasis. It has been shown that expression and activity of CFTR can be regulated either positively or negatively by PDZ (PSD-95/discs large/ZO-1) domain-based adaptors. Although a positive regulation by PDZ domain-based adaptors such as EBP50/NHERF1 is established, the mechanisms for negative regulation of the CFTR by Shank2, as well as the effects of multiple adaptor interactions, are not known. Here we demonstrate a physical and physiological competition between EBP50-CFTR and Shank2-CFTR associations and the dynamic regulation of CFTR activity by these positive and negative interactions using the surface plasmon resonance assays and consecutive patch clamp experiments. Furthermore whereas EBP50 recruits a cAMP-dependent protein kinase (PKA) complex to CFTR, Shank2 was found to be physically and functionally associated with the cyclic nucleotide phosphodiesterase PDE4D that precludes cAMP/PKA signals in epithelial cells and mouse brains. These findings strongly suggest that balanced interactions between the membrane transporter and multiple PDZ-based adaptors play a critical role in the homeostatic regulation of epithelial transport and possibly the membrane transport in other tissues. Disorganized ion transport caused by hypo- or hyperfunctioning of the cystic fibrosis transmembrane conductance regulator (CFTR) can be detrimental and may result in life-threatening diseases such as cystic fibrosis or secretory diarrhea. Thus, CFTR is controlled by elaborate positive and negative regulations for an efficient homeostasis. It has been shown that expression and activity of CFTR can be regulated either positively or negatively by PDZ (PSD-95/discs large/ZO-1) domain-based adaptors. Although a positive regulation by PDZ domain-based adaptors such as EBP50/NHERF1 is established, the mechanisms for negative regulation of the CFTR by Shank2, as well as the effects of multiple adaptor interactions, are not known. Here we demonstrate a physical and physiological competition between EBP50-CFTR and Shank2-CFTR associations and the dynamic regulation of CFTR activity by these positive and negative interactions using the surface plasmon resonance assays and consecutive patch clamp experiments. Furthermore whereas EBP50 recruits a cAMP-dependent protein kinase (PKA) complex to CFTR, Shank2 was found to be physically and functionally associated with the cyclic nucleotide phosphodiesterase PDE4D that precludes cAMP/PKA signals in epithelial cells and mouse brains. These findings strongly suggest that balanced interactions between the membrane transporter and multiple PDZ-based adaptors play a critical role in the homeostatic regulation of epithelial transport and possibly the membrane transport in other tissues. The cystic fibrosis transmembrane conductance regulator (CFTR) 2The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; AKAP, cAMP-dependent protein kinase-anchoring protein; IBMX, 3-isobutyl-1-methylxanthine; IP, immunoprecipitation; NHE, Na+/H+ exchanger; NTA, Ni2+-nitrilotriacetic acid; PDE, phosphodiesterase; PKA, cAMP-dependent protein kinase; PDZ, PSD-95/discs large/ZO-1; PSD, postsynaptic density; SPR, surface plasmon resonance; CHO, Chinese hamster ovary; GST, glutathione S-transferase; PR, proline-rich; UCR, upstream conserved region.2The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; AKAP, cAMP-dependent protein kinase-anchoring protein; IBMX, 3-isobutyl-1-methylxanthine; IP, immunoprecipitation; NHE, Na+/H+ exchanger; NTA, Ni2+-nitrilotriacetic acid; PDE, phosphodiesterase; PKA, cAMP-dependent protein kinase; PDZ, PSD-95/discs large/ZO-1; PSD, postsynaptic density; SPR, surface plasmon resonance; CHO, Chinese hamster ovary; GST, glutathione S-transferase; PR, proline-rich; UCR, upstream conserved region. is a Cl- channel and a key regulator of fluid and ion transport in the gastrointestinal, respiratory, and genitourinary epithelia (1Sheppard D.N. Welsh M.J. Physiol. Rev. 1999; 79: S23-S45Crossref PubMed Scopus (792) Google Scholar, 2Quinton P.M. Physiol. Rev. 1999; 79: S3-S22Crossref PubMed Scopus (307) Google Scholar). Hypofunctioning of CFTR due to genetic defects causes cystic fibrosis, the most common lethal genetic disease in Caucasians (3Choi J.Y. Muallem D. Kiselyov K. Lee M.G. Thomas P.J. Muallem S. Nature. 2001; 410: 94-97Crossref PubMed Scopus (338) Google Scholar, 4Anderson M.P. Rich D.P. Gregory R.J. Smith A.E. Welsh M.J. Science. 1991; 251: 679-682Crossref PubMed Scopus (430) Google Scholar), whereas hyperfunctioning of CFTR resulting from various infections evokes secretory diarrhea (5Chao A.C. de Sauvage F.J. Dong Y.J. Wagner J.A. Goeddel D.V. Gardner P. EMBO J. 1994; 13: 1065-1072Crossref PubMed Scopus (231) Google Scholar), the leading cause of mortality in early childhood in the world (World Health Organization statistics). Therefore, maintaining and regulating a dynamic balance between CFTR-activating and CFTR-inactivating machineries is an important mechanism for maintaining body homeostasis. Accumulating evidence suggests that protein-protein interactions play a critical role in the regulation of CFTR and other epithelial transporters (6Lamprecht G. Seidler U. Am. J. Physiol. 2006; 291: G766-G777Crossref PubMed Scopus (121) Google Scholar, 7Donowitz M. Cha B. Zachos N.C. Brett C.L. Sharma A. Tse C.M. Li X. J. Physiol. 2005; 567: 3-11Crossref PubMed Scopus (182) Google Scholar, 8Ahn W. Kim K.H. Lee J.A. Kim J.Y. Choi J.Y. Moe O.W. Milgram S.L. Muallem S. Lee M.G. J. Biol. Chem. 2001; 276: 17236-17243Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). PDZ (PSD-95/discs large/ZO-1) domain-based adaptors, best studied in the postsynaptic density (PSD) region of neurons, have emerged as a large group of proteins that sequester functionally related groups of transporters, receptors, and other effector proteins into integrated molecular complexes (9Funke L. Dakoji S. Bredt D.S. Annu. Rev. Biochem. 2005; 74: 219-245Crossref PubMed Scopus (366) Google Scholar, 10Kim E. Sheng M. Nat. Rev. Neurosci. 2004; 5: 771-781Crossref PubMed Scopus (1220) Google Scholar). Epithelial cells also utilize specific PDZ proteins to direct the polarized activities in their apical and basolateral membranes. Recently we reported functional and physical associations between the PDZ domain-containing protein Shank2 and two epithelial transporters, CFTR and the Na+/H+ exchanger 3 (NHE3) (11Han W. Kim K.H. Jo M.J. Lee J.H. Yang J. Doctor R.B. Moe O.W. Lee J. Kim E. Lee M.G. J. Biol. Chem. 2006; 281: 1461-1469Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 12Kim J.Y. Han W. Namkung W. Lee J.H. Kim K.H. Shin H. Kim E. Lee M.G. J. Biol. Chem. 2004; 279: 10389-10396Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Interestingly Shank2 attenuated the cAMP-dependent regulation of CFTR and NHE3. Conversely it has been shown that PDZ domain-based adaptors, such as EBP50/NHERF1 and E3KARP/NHERF2, can enhance the effects of cAMP on these transporters by recruiting a cAMP-dependent protein kinase (PKA)/PKA-anchoring protein (AKAP) complex (13Weinman E.J. Steplock D. Wang Y. Shenolikar S. J. Clin. Investig. 1995; 95: 2143-2149Crossref PubMed Scopus (311) Google Scholar, 14Yun C.H. Lamprecht G. Forster D.V. Sidor A. J. Biol. Chem. 1998; 273: 25856-25863Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Physiological significance of these adaptors in the regulation of CFTR function has also been verified in many other studies (6Lamprecht G. Seidler U. Am. J. Physiol. 2006; 291: G766-G777Crossref PubMed Scopus (121) Google Scholar). For example, recent evidence demonstrates that native CFTR and EBP50 are co-immunoprecipitated in human airway epithelial cells (15Taouil K. Hinnrasky J. Hologne C. Corlieu P. Klossek J.M. Puchelle E. J. Biol. Chem. 2003; 278: 17320-17327Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) and that CFTR-dependent anion current is reduced in the intestine of EBP50 knock-out mice (6Lamprecht G. Seidler U. Am. J. Physiol. 2006; 291: G766-G777Crossref PubMed Scopus (121) Google Scholar). The PDZ domain of Shank proteins has a three-dimensional structure very similar to the PDZ domains of EBP50/NHERF1 (16Im Y.J. Lee J.H. Park S.H. Park S.J. Rho S.H. Kang G.B. Kim E. Eom S.H. J. Biol. Chem. 2003; 278: 48099-48104Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). In particular, these PDZ domains all contain a negatively charged amino acid at the end of the β C strand of the PDZ domain structures (Glu43 in hEBP50, Asp634 in rShank1, and Asp80 in hShank2) that preferentially interacts with a positively charged residue at the -1 position in the C terminus of the membrane transporters, such as -TRL in CFTR. Therefore, the question arises whether EBP50 and Shank2 have distinct CFTR binding sites or mutually compete for binding of a single site in cells expressing both adaptor proteins. In the present study, we determined the kinetic properties and physiological significance of the interactions between CFTR and the PDZ-based adaptors EBP50 and Shank2. Using surface plasmon resonance (SPR) assays, we found that the dissociation constant (KD) of CFTR-Shank2 binding was similar to that of CFTR-EBP50 binding and that both proteins apparently compete for binding at the same site. Consecutive patch clamp studies revealed that CFTR Cl- channel activity was dynamically regulated by the competition of Shank2 and EBP50 binding. Notably in contrast to the PKA/AKAP recruitment by EBP50 (6Lamprecht G. Seidler U. Am. J. Physiol. 2006; 291: G766-G777Crossref PubMed Scopus (121) Google Scholar), Shank2 was found to tether PDE4D to the CFTR complex, thus attenuating cAMP/PKA signals. Our results suggest that the competitive balance between Shank2 and EBP50 binding to the CFTR channel may maintain homeostatic regulation of epithelial ion and fluid transport. Cell Cultures and Plasmid Vectors—NIH 3T3 and COS7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. T84 cells were purchased from the American Type Culture Collection (ATCC CCL-248) and maintained in a 1:1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum. CHO-K1 cells (KCLB 10061; Korea Cell Line Bank, Seoul, Korea) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. The pcDNA3.1-rShank2/CortBP1 (17Lim S. Naisbitt S. Yoon J. Hwang J.I. Suh P.G. Sheng M. Kim E. J. Biol. Chem. 1999; 274: 29510-29518Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), pcDNA3.1-hEBP50/NHERF-1 (12Kim J.Y. Han W. Namkung W. Lee J.H. Kim K.H. Shin H. Kim E. Lee M.G. J. Biol. Chem. 2004; 279: 10389-10396Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), and pCMV5 vectors containing rPDE4D1 to rPDE4D9 have been described previously (18Richter W. Jin S.L. Conti M. Biochem. J. 2005; 388: 803-811Crossref PubMed Scopus (120) Google Scholar). SPR Measurements and Kinetic Analysis of Sensorgrams— PDZ1-(1–139), PDZ2-(132–299), and PDZ1+2-(1–299) domains of hEBP50/NHERF1 and the PDZ domain of rShank2/CortBP1-(1–142) were PCR-amplified and cloned into the pRSET A vector (Invitrogen) to create The amino acid of and of competition were PCR-amplified and cloned into the vector to create were by proteins were in cells and on Ni2+-nitrilotriacetic acid The and as The proteins were using protein and their was as by of SPR with an was used to PDZ proteins. at various in were at a of The was between using of containing and were by the by the were for by the by on and kinetic were by the of Cl- were on CHO-K1 cells as reported previously W. Kim K.H. Lee M.G. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The and and the and were at the CFTR was by The used was and the current was at were and using an and a channel activity of CFTR was in using with a of The and and the and patch were by the of and channel EBP50 at was to the used in the single channel was The was used for and The and current were at the and the single channel were at The of in a patch was determined from the of at of was to the phosphodiesterase in 3T3 cells and T84 cells using reported previously (12Kim J.Y. Han W. Namkung W. Lee J.H. Kim K.H. Shin H. Kim E. Lee M.G. J. Biol. Chem. 2004; 279: 10389-10396Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The specific to and were from common to both mouse and human C and and T84 and were with and containing mixture were by and were with the and at complexes were by for at with protein and with to The and were in and for and by The proteins were to and the were by for in a containing 5% in and The were with the and and protein were with (17Lim S. Naisbitt S. Yoon J. Hwang J.I. Suh P.G. Sheng M. Kim E. J. Biol. Chem. 1999; 274: 29510-29518Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar) and (18Richter W. Jin S.L. Conti M. Biochem. J. 2005; 388: 803-811Crossref PubMed Scopus (120) Google Scholar) and the mouse S. M. C. M. Conti M. 1998; PubMed Scopus Google Scholar) were described was purchased from of was as reported previously (12Kim J.Y. Han W. Namkung W. Lee J.H. Kim K.H. Shin H. Kim E. Lee M.G. J. Biol. Chem. 2004; 279: 10389-10396Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). from were in in and into The were by with and by with were with a to of and domains were PCR-amplified and cloned into vector to create Shank2 domains were and two were and proteins were in E. and with For assays, cells were on in a containing and was by and of were supplemented with to of of at were supplemented with of and for an at The was and at with containing and to in and Measurements of from and PDE4D knock-out mice S.L. F.J. Conti M. U. S. A. 1999; PubMed Scopus Google Scholar) were in containing 10% and a mixture of Cell were at and were using of to of protein for were and activity was as described previously (18Richter W. Jin S.L. Conti M. Biochem. J. 2005; 388: 803-811Crossref PubMed Scopus (120) Google Scholar). In were in a mixture of containing bovine and of for at The was by the of of in by in a for The was by of the mixture with of for at The resulting was by anion using of and by Shank2 and EBP50 for on the of CFTR positive and negative we determined the binding of the between CFTR and the PDZ domains of EBP50 and Shank2 using SPR EBP50 has two PDZ PDZ1-(1–139), PDZ2-(132–299), and were used to their PDZ domains were on and their binding to the C terminus of CFTR was by shown in the the PDZ domains of Shank2 and EBP50 the C terminus of CFTR in a A and The dissociation at the of Shank2 PDZ and EBP50 were at the of Interestingly EBP50 has a of and dissociation Shank2 of EBP50 using and proteins revealed that the EBP50 binding to CFTR is by has a A competition was to the of competition between the of Shank2 and EBP50 in binding to CFTR. Shank2 PDZ on the SPR was with a of the C terminus of CFTR and of EBP50 the of EBP50 the between Shank2 PDZ and CFTR with an of of CFTR Cl- by CFTR-EBP50 and CFTR-Shank2 results that Shank2 and EBP50 compete for binding on CFTR. competitive balance may CFTR ion activities at the apical membrane of and Shank2 and EBP50 are M. Cha B. Zachos N.C. Brett C.L. Sharma A. Tse C.M. Li X. J. Physiol. 2005; 567: 3-11Crossref PubMed Scopus (182) Google Scholar, 12Kim J.Y. Han W. Namkung W. Lee J.H. Kim K.H. Shin H. Kim E. Lee M.G. J. Biol. Chem. 2004; 279: 10389-10396Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). determined the effects of the competition between EBP50 and Shank2 PDZ binding on the CFTR Cl- channel activity using patch from patch were with a for to CFTR was by the of the of and to the an ion channel activity with a single channel conductance of and a was that was in It has been shown that adaptors with multiple PDZ such as EBP50 and can CFTR Cl- channel activity of cAMP signals by protein U. S. A. 2001; PubMed Scopus Google Scholar, S. H. R.B. Li M. Full Text Full Text PDF PubMed Scopus Google Scholar). In with these with EBP50 a in the of CFTR. of Shank2 PDZ the in CFTR by A and The of Shank2 PDZ EBP50 competition was in Shank2 PDZ not in CFTR at it the CFTR by of EBP50 a in CFTR Shank2 and the using EBP50 not due to the of the we results similar to using the EBP50 protein C and These findings that Shank2 can the CFTR by with EBP50 for binding at the C terminus of CFTR and by the of CFTR. not the that Shank2 the and of CFTR in In 3T3 it has been shown that Shank2 CFTR channel activity and by the (12Kim J.Y. Han W. Namkung W. Lee J.H. Kim K.H. Shin H. Kim E. Lee M.G. J. Biol. Chem. 2004; 279: 10389-10396Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). It is well that CFTR is by of the by P.M. Physiol. Rev. 1999; 79: S3-S22Crossref PubMed Scopus (307) Google Scholar). by Shank2 of cyclic nucleotide cAMP or the of PKA, these The is CFTR was in the cells in the of a of the (12Kim J.Y. Han W. Namkung W. Lee J.H. Kim K.H. Shin H. Kim E. Lee M.G. J. Biol. Chem. 2004; 279: 10389-10396Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Shank2 with important was in an with the of cells with 3 a CFTR Cl- current that is on the of Cl- in and is by and has in Shank2 was the same of very or CFTR Interestingly with a CFTR current in was that of cells A and These results suggest a functional between the activity and the of CFTR. the and determined whether Shank2 interacts with the and are for Shank2 as are to and are to be in epithelial cells C. M. Jin S.L. Conti M. 13: Full Text Full Text PDF PubMed Scopus Google Scholar). The of two and and the of results using specific to the common of mouse and human revealed that and and are in mouse 3T3 cells and human T84 cells both and are the patch clamp shown in was of the cells with the and the C and not the effects of Shank2 Conversely was by 3 a CFTR current in cells strongly an between Shank2 and Recently two that PDE4D a role in a cAMP at the apical of epithelial cells G. P. C. Conti M. M.J. Milgram S.L. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. L. A. E. C. D. J. 2005; PubMed Scopus Google Scholar). with expression of PDE4D proteins was in the of CHO, and T84 cells in the effects of Shank2 on cAMP/PKA signals were in and studies (11Han W. Kim K.H. Jo M.J. Lee J.H. Yang J. Doctor R.B. Moe O.W. Lee J. Kim E. Lee M.G. J. Biol. Chem. 2006; 281: 1461-1469Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 12Kim J.Y. Han W. Namkung W. Lee J.H. Kim K.H. Shin H. Kim E. Lee M.G. J. Biol. Chem. 2004; 279: 10389-10396Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). for a physical between Shank2 and were and PDE4D have multiple that for at to (18Richter W. Jin S.L. Conti M. Biochem. J. 2005; 388: 803-811Crossref PubMed Scopus (120) Google Scholar). has been shown to be in epithelial cells and to cAMP at the apical G. P. C. Conti M. M.J. Milgram S.L. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, Shank2 and the PDE4D were in cells by with Shank2 and Shank2 proteins were in with In a be in Shank2 These were specific as be in cells expressing both Shank2 and Shank2 were and used for Shank2 has multiple sites for protein-protein interactions, a PDZ a and a domain M. Kim E. J. Cell PubMed Google Scholar). The region a domain J. J. PubMed Scopus Google Scholar). In assays using the from the an with the region of Shank2 The region was into two and and the PDE4D binding site was to the region and a are all PDE4D were co-immunoprecipitated with Shank2 to the PDE4D that with Shank2 and to the Shank2 binding site in cells were with Shank2 and the PDE4D to by using the PDE4D and were found to with Shank2, whereas the and not A was in the of The PDE4D are by the or of two conserved domains upstream conserved and and The results that and the of Shank2 binding. Shank2 and PDE4D in between Shank2 and PDE4D was in cells that both proteins to physiological we the expression of Shank2 and PDE4D in Shank2 and PDE4D were at the apical region of CFTR a role in fluid and ion The was also verified by of the proteins from T84 human epithelial It is well that can be by of M. W. C. G. Park J.Y. Jin C. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). Interestingly of the cAMP/PKA using the the physical between Shank2 and PDE4D in T84 cells the was in mouse multiple as well as Shank2 are at In using multiple were in to the Shank2 due to the of distinct Shank2 in (17Lim S. Naisbitt S. Yoon J. Hwang J.I. Suh P.G. Sheng M. Kim E. J. Biol. Chem. 1999; 274: 29510-29518Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, J. J. PubMed Scopus Google Scholar). was with and the resulting were to activity The activity with Shank2 was by the In the of was in using from PDE4D knock-out mice that Shank2 interacts with PDE4D in mouse In the present study, we evidence that the activity of CFTR is dynamically regulated by a competitive balance between CFTR-activating and CFTR-inactivating PDZ domain In activity of Shank2, the CFTR-inactivating PDZ was found to be associated with precludes cAMP/PKA signals. The mechanisms resulting from the of CFTR with EBP50 and Shank2 are in It is that EBP50 can CFTR two EBP50 can a and the of CFTR M. Cha B. Zachos N.C. Brett C.L. Sharma A. Tse C.M. Li X. J. Physiol. 2005; 567: 3-11Crossref PubMed Scopus (182) Google Scholar). EBP50 can CFTR by possibly by a CFTR U. S. A. 2001; PubMed Scopus Google Scholar). The results of revealed that Shank2 can both mechanisms by with EBP50 at the C terminus of CFTR. The that Shank2 the of CFTR in that Shank2 can the EBP50 function by the of CFTR of cAMP signals. that Shank2 can cAMP in the of the CFTR by PDE4D into It is well that cAMP signals are in many and that cAMP signals play a critical role in various physiological M. W. C. G. Park J.Y. Jin C. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). For example, cAMP in a region with the and the in is for the regulation of by M. Science. PubMed Scopus Google Scholar). recent the of PDE4D as a cAMP at the apical membrane of epithelia for the of CFTR G. P. C. Conti M. M.J. Milgram S.L. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. L. A. E. C. D. J. 2005; PubMed Scopus Google Scholar). the molecular mechanisms for in apical A molecular in revealed that the apical adaptor Shank2 recruits PDE4D a direct between the region of Shank2 and the region of The of cAMP regulating an in regulating CFTR Recently a of Shank2 with and a 3 was in epithelial cells E. L. Doctor R.B. Biochem. J. 2004; PubMed Scopus Google Scholar). Therefore, we the in 3 with The results were to using Shank2 that domains the structure of Shank2 are important for regulating CFTR function not The physiological significance of the is not to epithelial cells can be to other For example, Shank2 is in many of the and Shank proteins are the key of and with M. Kim E. J. Cell PubMed Google Scholar, B. Naisbitt S. P. A. Sheng M. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), is for efficient of that play a critical role in and of are not associated with also with cAMP and it is that cAMP signals play a role in the functional of A. U. S. A. PubMed Scopus Google Scholar, M. Y. U. S. A. 2006; PubMed Scopus Google Scholar). Therefore, PDE4D may play a critical role in the regulation of in studies with knock-out of PDZ proteins and have shown that the of these adaptors are complex previously and to be (6Lamprecht G. Seidler U. Am. J. Physiol. 2006; 291: G766-G777Crossref PubMed Scopus (121) Google Scholar). PDZ domains have well binding are for as the present is to be the function of PDZ-based adaptor PDZ interactions can a Nat. Rev. 2004; PubMed Scopus Google Scholar). For example, as the PDZ structures of EBP50 and Shank2 are very or EBP50 may have effects in by Shank2 complexes as In the present demonstrates the functional of protein-protein interactions and that signals can be to the same of a membrane protein by adaptors. A competitive balance between the and PDZ interactions be critical in the regulation of many membrane transporters and as for CFTR. S. Muallem at the of and S. L. Milgram at the of for and the of the and for on the