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1 Dynamic regulation of CFTR chloride channel activity by PDZbased scaffolds Ji Hyun Lee Department of Medical Science The Graduate School, Yonsei University

2 Dynamic regulation of CFTR chloride channel activity by PDZbased scaffolds Directed by Professor Min Goo Lee The Doctoral Dissertation submitted to the Department of Medical Science, the Graduate School of Yonsei University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Ji Hyun Lee December 2007

3 The certifies that the Doctoral Dissertation of Ji Hyun Lee is approved Thesis Supervisor: Min Goo Lee Thesis Committee Member: Jeung-Gweon Lee Thesis Committee Member: Joo-Heon Yoon Thesis Committee Member: Man Wook Hur Thesis Committee Member: Dong-Min Shin The Graduate School Yonsei University December 2007

4 ACKNOWLEDGEMENTS 제가이곳약리학교실과인연을맺은지벌써 6년이란시간이흘렀습니다. 학부 4학년때유학을갈지아니면취직을할지아직무엇을해야할지정하지못해서고민하고있을때우연히남궁완선배를따라서이곳약리학교실에왔다가지금제지도교수님이신이민구교수님께인사만드리려고했는데 언제부터나올수있지? 라는한마디에 4학년 2학기때부터학부수업을마치면곧장이곳약리학교실에와서실험을배우면서자연스럽게석사과정에입학하였습니다. 힘들었지만참즐거웠던 2년의석사과정을마치고여기서박사를할지아니면유학을갈지또한번고민을할때이민구교수님께서이렇게말씀을하셨습니다. 석사만하면진정한선생님학생이아니고박사를해야진정한선생님학생이다 라고. 이렇게저는또이곳약리학교실박사과정에입학하였고 4년이란시간동안의제연구결과를박사학위논문으로정리하려고합니다. 돌이켜보면이곳약리학교실에서 6년이란시간은제 20대의반을보낸길고도짧은시간이었고, 주위에많은고마운분들이있었기에지금의제가있을수있었습니다. 제일먼저제지도교수님이신이민구교수님께감사하다는말을전하고싶습니다. 워낙말이없으셔서처음에는너무나어려운교수님이셨지만처음부터저와의인연을소중히생각하셨고저에게과학자로서의사고방식과마음가짐을가르쳐주신참좋으신분이셨습니다. 그리고지금은우리곁을떠났지만친할아버지처럼따뜻하게반겨주셨던이우주교수님, 항상존재감만으로도너무나도든든하고더열심히연구하라고가끔맛난것도사주시는김경환교수님, 지금대회협력본부장이셔서너무나도바쁘시고또마라톤을좋아하시는멋진안영수교수님, 우리교실주임교수님이시고항상웃는얼굴로따뜻하게대하여주시는김동구교수님, 멋진이력을갖고계시고현재우리학교임상시험센터를이끌고계신박경수교수님, 교수님이시지만아직도가방을

5 메고다니셔서젊음과열정이느껴지는김철훈교수님, 그리고지금은본교식품영양학과로자리를옮기셨지만항상세심하게챙겨주셨던김혜영교수님, 처음엔선배로인연을시작하였지만지금은교수님이되셔서나의본보기가되는김주영교수님, 언제나아침일찍오셔서열심히연구하는열정적인연구자의모습을보여주신임주헌교수님께진심으로감사드립니다. 또한아직많이부족한저에게자문기간동안항상많이칭찬해주셨던이정권교수님, 그리고자문위원이시기이전부터저에게많은격려와관심을가져주셨던윤주헌교수님, 보다좋은학위논문이될수있도록학위논문을꼼꼼히검토해주시고많은자문을해주신허만욱교수님, 자문기간동안바쁘신데도항상제일먼저챙겨주신신동민교수님께도진심으로감사드립니다. 즐겁고힘들었던시간을늘함께한실험실식구들저에게는참든든한동반자였습니다. 나와이곳약리학교실과의인연을만들어주었고언제나나에게따뜻했던완선배, 아직도가끔전화해서실험실소식을묻는민재오빠, 아직많은것을모르는나에게항상충고를아끼지않는우인선배, 항상즐거운이야기로우리를웃겼던써니언니, 선배로써좀더잘해주지못해서항상미안한요셉이와승근이모두이곳약리학교실을떠났지만진심으로감사하다는말을전합니다. 오빠라고부르면서좀더친하게지내고싶었지만그러지못해서너무나도아쉬운이성희선생님, 매사너무나도열심히생활해서항상나를돌아보게만드는헌영오빠, 후배이지만가끔은나의고민을들어주는든든한현우, 넓은마음을가졌고우리연구실안주인정남누나, 항상매사열심히하고지금치프로서우리랩을이끌고있는재석이, 성격이쿨한이정수선생님, 우리랩막내우영이, 포스트닥으로새롭게우리랩에합류한곽진오선생님께도감사합니다. 그리고내가아무리괴롭혀도항상웃는얼굴로잘설명해주셨던이진우선생님, 아니지금은이진우교수님, 언제나열심히연구하는이정호선생님, 고향이같아서친근한기호, 나를너무나도잘따라서친동생같은우리, 5

6 예쁘고터프한재현이, 요즘실험이많아서조금힘들어보이는미경이, 조용조용열심히하는영신이, 참성격이좋고열심히하는복이, 좀더잘먹고살이쪄야하는이쁜이효선이, 그리고임상시험센터에있어서많이볼수없어서아쉬운윤정이와아영선생님, 항상따뜻한말을건네는주경돈선생님, 잠시떠났다가다시교실로돌아온순옥이와동휘, 지금본교식품영양학과로자리를옮겨서자주볼수없어서아쉬운정혜연선생님과서정연선생님.. 모두감사합니다. 그리고이번에같이졸업하는인숙언니, 이장원선생님, 보람이모두수고많았습니다. 언제나약리학교실을지켜주시고참순수하고따뜻한마음을가진선해언니, 맥가이버처럼무슨일이든척척해결해주시는멋쟁이임종수선생님, 그리고김건태선생님께도감사의말을전합니다. 힘들고지칠때위로가되어준네소중한친구홍은이, 희원이, 드디어좋은짝은만나곧장가가는나를가장잘이해해주었고챙겨주었던친구정효, 그리고 3년동안기숙사생활을같이해서항상편하고든든한내고등학교동기들, 후배이지만항상나를챙겨준세희, 항상힘내라고맛난것을사준정현선배, 그리고후배민희, 용식이에게진심으로감사의마음을전합니다. 끝으로지금까지저를키워주시고늘마음속의큰나무가되어주시는부모님께진심으로감사드립니다. 그리고누구보다도나를아껴주고같이다니면가끔남자친구로오인받는하나밖에없는우리오빠에게도감사의마음을전합니다. 6

7 TABLE OF CONTENTS ABSTRACT I. INTRODUCTION II. MATERIALS AND METHODS Cell Cultures and Plasmid Vectors Surface Plasmon Resonance (SPR) Measurements and Kinetic Analysis of Sensorgrams Measurements of Cl - Channel Activities Reverse Transcription-PCR Immunoprecipitation and Immunoblotting GST Pulldown Assay Immunohistochemistry Measurements of PDE Activity III. RESULTS Shank2 and EBP50 Compete for Binding on CFTR Regulation of CFTR Cl - Channel Activity by CFTR-EBP50 and CFTR-Shank2 Competitions i

8 3. Shank2 Associates with PDE4D Shank2 and PDE4D Associate in Vivo IV. DISCUSSION V. CONCLUSION REFERENCES ABSTRACT (IN KOREA) PUBLICATION LIST ii

9 LIST OF FIGURES FIGURE 1. Interactions between CFTR and PDZ-based adaptors determined by surface plasmon resonance (SPR) assays FIGURE 2. Regulation of CFTR single channel activity by a competition between CFTR-EBP50 and CFTR- Shank2 PDZ binding FIGURE 3. Shank2 associates with PDE4D FIGURE 4. Domains responsible for the association between Shank2 and PDE4D FIGURE 5. Shank2 and PDE4D associate in vivo FIGURE 6. A model for the regulation of CFTR through interaction with EBP50 and Shank iii

10 ABSTRACT Dynamic regulation of CFTR chloride channel activity by PDZ-based scaffolds Ji Hyun Lee Department of Medical Science The Graduate School, Yonsei University (Directed by Professor Min Goo Lee) Disorganized ion transport caused by hypo- or hyper-functioning 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. Accordingly, 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 scaffolds. Although a positive regulation by PDZ domain-based scaffolds such as EBP50/NHERF1 is established, the mechanisms for negative regulation of the CFTR by Shank2, as well as the effects of multiple scaffold interactions, are not known. Therefore, I demonstrated 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 - 1 -

11 assays and 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 scaffolds play a critical role in the homeostatic regulation of epithelial transport and possibly the membrane transport in other tissues. Key Word: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), PDZ-based scaffolds, EBP50, Shank2, PDE4, camp - 2 -

12 Dynamic regulation of CFTR chloride channel activity by PDZ-based scaffolds Ji Hyun Lee Department of Medical Science The Graduate School, Yonsei University (Directed by Professor Min Goo Lee) I. INTRODUCTION CFTR is a Cl - channel and the key regulator of fluid and ion transport in the gastrointestinal, respiratory, and genitourinary epithelia 1. Hypofunctioning of CFTR due to genetic defects causes cystic fibrosis, the most common lethal genetic disease in Caucasians 2,3, whereas hyper-functioning of CFTR resulting from various infections evokes secretory diarrhea 4, the world s leading cause of mortality in early childhood ( Therefore, maintaining and regulating a dynamic balance between CFTRactivating 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 5,6. PDZ (PSD-95/discs large/zo-1)-based scaffolds, best studied in the post-synaptic density (PSD) region of neurons, have emerged as a large group of proteins that sequester functionally-related groups of transporters, receptors, and other - 3 -

13 effector proteins into integrated molecular complexes 7. Epithelial cells also utilize specific PDZ proteins to direct the polarized activities in their apical and basolateral membranes. Previous reports described that functional and physical associations between the PDZ domain-containing protein Shank2 and two epithelial transporters, the cystic fibrosis transmembrane conductance regulator (CFTR) and the Na + /H + exchanger 3 (NHE3) 8,9. Interestingly, Shank2 attenuated the camp-dependent regulation of CFTR and NHE3. Conversely, it has been shown that PDZ-based scaffolds, such as EBP50/NHERF1 and E3KARP/NHERF2, can enhance the effects of camp on these transporters by recruiting a camp-dependent protein kinase anchoring protein (AKAP)/PKA complex 10,11. The PDZ domain of Shank proteins has a three-dimensional structure very similar compared to the PDZ domains of EBP50/NHERF1. In particular, these PDZ domains all contain a negatively-charged amino acid at the end of the βc strand of the PDZ domain structures (Glu 43 in hebp50, Asp 634 in rshank1, and Asp 80 in hshank2) 12, which 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, I determined the kinetic properties and physiological significance of the interactions between CFTR and the PDZ-based adaptors, EBP50 and Shank

14 II. MATERIALS AND METHODS 1. 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 13, pcdna3.1-hebp50/nherf-1 8, and pcmv5 vectors containing rpde4d1 to rpde4d9 have been described previously Surface Plasmon Resonance (SPR) Measurements and Kinetic Analysis of Sensorgrams PDZ1 (1-139), PDZ2 ( ), 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 (Life Technologies Inc., Gaithersburg, MD) to create His-tagged constructs. C-terminal 29 aa residues of hcftr (NM_000492) and PDZ1+2 of hebp50 were PCR-amplified and cloned into the pgex-4t-1 vector (Amersham Biosciences, Piscataway, NJ) to create GST-fusion constructs. All constructs were confirmed by sequence analysis. All fusion proteins were expressed in E. coli BL21 (DE3) cells and purified on Ni 2+ -nitrilotriacetic acid (NTA) resin (Qiagen, Venlo, The Netherlands) and glutathione-sepharose 4B (Amersham Biosciences), as appropriate. The - 5 -

15 purified proteins were quantified using Bio-Rad protein assay reagent, and their purity was assessed as >90% by Coomassie staining of SDS-PAGE gels. An SPR system equipped with an NTA chip (BIAcore 3000; Biacore AB, Uppsala, Sweden) was used to capture His-tagged PDZ fusion proteins. Analytes (GSTtagged proteins) at various concentrations in HEPES-buffered saline-ep (0.01 M Hepes, 0.15 M NaCl, 50 µm EDTA, 0.005% Tween20, ph 7.4) were perfused at a flow rate of 30 µl/min. The sensor chip was regenerated between each analysis using successive injections of solutions containing 0.15 M NaCl and 0.35 M EDTA. Response curves were generated by subtracting the background signal generated simultaneously by the control flow cell. Background-subtracted curves were prepared for fitting by subtracting the signal generated by buffer alone on experimental flow cells. Sensorgram curves and kinetic parameters were evaluated by the BIAEVALUATION 3.1 software (Biacore AB), which uses numerical integration algorithms. 3. Measurements of Cl - Channel Activities Whole-cell recordings were performed on CFTR transfected CHO-K1 cells. The pipette solution contained (in mm) 140 N-methyl D-glucamine chloride (NMDG-Cl), 5 EGTA, 1 MgCl 2, 1 Tris-ATP, and 10 HEPES (ph 7.2), and the bath solution contained 140 NMDG-Cl, 1 CaCl 2, 1 MgCl 2, 10 Glucose, and 10 HEPES (ph 7.4). All experiments were performed at room temperature (22-25 C). Pipettes were pulled from borosilicate glass and had resistances of 3-5 MΩ after fire polishing. Seal resistances were typically between 3-10 GΩ. After establishing the whole-cell configuration, CFTR was activated by adding - 6 -

16 forskolin and/or IBMX. The holding potential used was -30 mv and the current output was filtered at 5 khz. Currents were digitized and analyzed using an AxoScope 8.1 system and a Digidata 1322A AC/DC converter (Axon Instruments, Union City, CA). Single channel activity of CFTR was measured in inside-out configurations using fire-polished pipettes with a resistance of MΩ. The pipette solution contained (in mm) 140 NaCl, 5 KCl, 1 MgCl 2, 1 CaCl 2, and 10 HEPES (ph 7.2), and the bath solution contained 140 NaCl, 5 KCl, 1 MgCl 2, and 10 HEPES (ph 7.4). Following patch excision, channels were activated by adding the catalytic subunit of PKA (40 unit/ml; Promega, Madison, WI) and 1 mm MgATP. After channel activation, EBP50 and/or Shank2-PDZ at desired concentrations were added to the bath. Holding voltage used in the single channel recording was +60 mv. The pclamp software package (version 9.2, Axon Instruments) was used for data acquisition and analysis. The voltage and current data were low-pass filtered at 1 khz during the recordings and the single channel data were further digitally filtered at 25 Hz. Channel open probability (P o ) was estimated using the following equation: N P o = ti / TN i= l where t i is the time spent above a threshold set at 0.5 times channel current amplitude, T is the duration of the recording, and N is the number of channels in patch. The number of active channels in a patch was determined from the number of simultaneously open channels during at least 15 min of recording

17 4. Reverse Transcription-PCR RT-PCR analysis was performed to identify the PDE isoforms in NIH 3T3 cells and T84 cells. The primer sequences specific to PDE3 and PDE4 were selected from regions common to both mouse and human PDEs: 1) PDE3A, sense (5 -CAC AGG GCC TTA ACT TAC AC-3 ), antisense (5 -TTG AGT CCA GGT TAT CCA TGA C-3 ), PCR product 370 bp; 2) PDE3B, sense (5 - CAG GAA GGA TTC TCA GTC AGG-3 ), antisense (5 -GTC ATT GTA TAA AAC TGC CTG AGG-3 ), PCR product 464 bp; 3) PDE4A, sense (5 -ATC AAC ACC AAT TCG GAG C C-3 ), antisense (5 -TCA CCC TGC TGG AAG AAC TC-3 ), PCR product 398 bp; 4) PDE4B, sense (5 -AGT CCT TGG AAT TGT ATC GG-3 ), antisense (5 -CTG GAT CAA TCA CAC AAA GCG TC-3 ), PCR product 432 bp; 5)PDE4C, sense (5 -TTC CAG ATC CCA GCA GAC AC- 3 ), antisense (5 -ATG ACC ATC CTG CGC AGA CTC-3 ), PCR product 392 bp; 6) PDE4D, sense (5 -GCC AAG GAA CTA GAA GAT GTG-3 ), antisense (5 -CAT CAT GTA TTG CAC TGG C-3 ), PCR product 328 bp. 5. Immunoprecipitation and Immunoblotting CHO-K1, COS-7, T84 cells, and rat cerebellum were lysed with lysis buffer (20 mm HEPES ph 7.4, 200 mm NaCl, 5 mm EDTA, 1% Triton X-100, 1 mm NaVO4, 1 mm β-glycerophosphate) containing complete protease inhibitor mixture (Roche Applied Science, Mannheim, Germany). After lysis, cell debris was removed by centrifugation, and cleared lysates were mixed with the appropriate antibodies and incubated overnight at 4 C. Immune complexes were collected by incubation for 2 h at 4 C with protein A/G PLUS agarose and - 8 -

18 washed four times with lysis buffer prior to electrophoresis. The immunoprecipitates and cell lysates were suspended in 2x SDS sample buffer and boiled for 5 min and then separated by SDS-PAGE. The separated proteins were transferred to nitrocellulose membranes, and the membranes were blocked by incubation for 1 h in a solution containing 5% nonfat dry milk in 20 mm Tris-HCl, ph 7.5, 150 mm NaCl, and 0.05% Tween 20. The membranes were then incubated with the appropriate primary and secondary antibodies, and protein bands were detected with enhanced chemiluminescence solutions. Rabbit polyclonal antibodies anti-shank2 1136, anti-pde4d5 α-4d5, and the mouse monoclonal PAN-PDE4D antibody, M3S1 were described previously. Rabbit polyclonal PAN-PDE4D antibody, ab14613, was purchased from Abcam Inc., Cambridge, UK. 6. GST Pulldown Assay Regions corresponding to residues of rshank2 PDZ (1-142), PR ( ), and SAM ( ) domains were PCR-amplified and cloned into pgex-4t-1 vector (Amersham Biosciences) to create GST-fusion constructs. Shank2 PR domains were further dissected and two GST-fusion constructs were generated; PR1 ( ) and PR2 ( ). All fusion proteins were expressed in E. coli BL21 (DE3) and purified with glutathione-sepharose 4B (Amersham Biosciences). For pull-down assays, CHO cells were lysed on ice in a 1% Triton X-100 buffer containing 150 mm NaCl, 2 mm MgCl 2, 2 mm CaCl 2, 10 mm HEPES ph 7.4 and proteinase inhibitors (CompleteMini, Roche). Debris was removed by centrifugation and 100 µl of lysate supernatants was - 9 -

19 supplemented with 900 µl of buffer prior to addition of 10 µg of each GST fusion protein. Following overnight incubation at 4 C, samples were supplemented with 80 µl of glutathione Sepharose (Amersham Biosciences) and incubated for an additional 4 h at 4 C. The glutathione Sepharose was pelleted and washed (3 x 5 min) at 4 C with wash buffer (PBS containing 0.1% Triton X-100 and 100 mm β-mercaptoethanol) prior to resuspension in SDS sample buffer and SDS PAGE. 7. Immunohistochemistry Colon tissues from Sprague-Dawley rats were embedded in OCT (Miles, Elkhart, IN), frozen in liquid N 2, and cut into 4-µm sections. The sections were fixed and permeabilized by incubation in cold methanol for 10 min at 20 C. Nonspecific binding sites were blocked by incubation for 1 h at room temperature with 0.1 ml of phosphate-buffered saline containing 5% goat serum, 1% bovine serum albumin, and 0.1% gelatin (blocking medium). After blocking, the sections were stained by incubation with polyclonal anti-shank and monoclonal anti-pan PDE4D M3S1 antibodies, followed by appropriate secondary antibodies tagged with fluorophores. Images were obtained with a Zeiss LSM 510 confocal microscope. 8. Measurements of PDE Activity Whole brain tissues from wild type and PDE4D knock-out mice were lysed in buffer containing 50 mm HEPES, ph 7.4, 250 mm NaCl, 10% glycerol, 0.5% Nonidet P-40, 1 mm EDTA, 0.2 mm EGTA, 10 m NaF, 10 mm Na 4 P 2 O 7,

20 1 µm microcystin, 2 mm 4-(2-aminoethyl) benzenesulfonylfluoride, and a mixture of protease inhibitors. Cell lysates were spun at 14,000 x g, and supernatants were immunoprecipitated using 5 µl of anti-shank2 antibody coupled to 25 µl of protein G sepharose for 4 h. After centrifugation, pellets were washed three times and PDE activity was then measured. In brief, samples were assayed in a reaction mixture of 200 µl containing 40 mm Tris-HCl, ph 8.0, 10 mm MgCl 2, 5 mm β-mercaptoethanol, 1 µm camp, 0.75 mg/ml bovine serum albumin, and 0.1 µciof [ 3 H]cAMP for 10 min at 33 C. The reaction was terminated by the addition of 200 µl of 10 mm EDTA in 40 mm Tris-HCl, ph 8.0, followed by heat inactivation in a boiling water bath for 1 min. The PDE reaction product 5'-AMP was then hydrolyzed by incubation of the assay mixture with 50 µg of Crotalus atrox snake venom for 20 min at 33 C. The resulting adenosine was separated by anion exchange chromatography using 1 ml of AG1-X8 resin and quantitated by scintillation counting

21 III. RESULTS 1. Shank2 and EBP50 Compete for Binding on CFTR To understand the dynamics of CFTR positive and negative regulations, I determined the binding kinetics of the association between CFTR and the PDZ domains of EBP50 and Shank2 using SPR analyses (FIG. 1). As EBP50 has two PDZ domains, three His-tagged proteins, PDZ1 (1-139), PDZ2 ( ), and PDZ1+2 (1-299), were used in order to analyze their kinetics separately. Histagged PDZ domains were captured on NTA chips, and their binding to the GST-tagged C-terminus of CFTR was sensored by SPR. As shown in the sensorgrams, the PDZ domains of Shank2 and EBP50 specifically bind the C- terminus of CFTR in a dose-dependent fashion (FIG. 1, a and b). The overall dissociation constants at the equilibrium state (KDs) of Shank2 PDZ and EBP50 PDZ1+2 were within comparable ranges at the tens of nanomolar levels (FIG. 1c). Interestingly, EBP50 PDZ1+2 has approximately a one order of magnitude faster association (Ka) and dissociation (Kd) kinetics than Shank2 PDZ. Further analyses of EBP50 PDZs using separate PDZ1 and PDZ2 proteins revealed that the overall EBP50 binding to CFTR is driven by PDZ1, which has a ~3 times higher affinity than PDZ2 (FIG. 1c). A solution competition assay was performed to examine the possibility of mutual competition between the PDZs of Shank2 and EBP50 in binding to CFTR. After capturing His-tagged Shank2 PDZ on NTA chips, the SPR system was perfused with a fixed dose of the C- terminus of CFTR (300 nm) and increasing doses of GST-tagged EBP50 PDZ1+2. Importantly, the addition of EBP50 PDZs dose dependently decreased the association between Shank2 PDZ and CFTR with an IC50 value of 91nM (FIG. 1d)

22 a CFTR C-term b CFTR C-term Response Difference (Resonance Unit) Shank2 PDZ CFTR C-term concentration 600 nm 400 nm 200 nm 100 nm 50 nm 0 nm Response Difference (Resonance Unit) EBP50 PDZ1+2 CFTR C-term concentration 160 nm 80 nm 40 nm 20 nm 10 nm 0 nm Time (S) Time (S) c d CFTR C-term (300 nm) EBP50 PDZ1+2 (variable conc.) Ligand Shank2 PDZ EBP50 PDZ1+2 EBP50 PDZ1 EBP50 PDZ2 Analyte CFTR C-term CFTR C-term CFTR C-term CFTR C-term Ka (M -1 s -1 ) 3.07x x x x10 4 Kd (s -1 ) 1.73x x x x10-3 KD (Kd/Ka, M) 5.63x x x x10-8 Response Difference (Resonance Unit) Shank2 PDZ Time (S) EBP50 PDZ1+2 concentration 0 nm 5 nm 10 nm 15 nm 30 nm 60 nm 100 nm 150 nm 200 nm 300 nm FIGURE 1. Interactions between CFTR and PDZ-based adaptors determined by surface plasmon resonance (SPR) assays (a, b) Sensorgrams showing the interaction between the C-terminus of CFTR and the PDZ domains of Shank2 and EBP50/NHERF1. His-tagged PDZ domains of Shank2 and EBP50 were captured on nitrilotriacetic acid (NTA) chips which were then perfused with various concentrations of a peptide encoding the C-terminal 29 aa of CFTR. (c) Binding kinetics of the interaction between CFTR and the PDZ domains of Shank2 and EBP50. Values shown represent the average of two experiments each performed in duplicate. (d) Sensorgram of a solution competition assay between CFTR-Shank2 PDZ and CFTR-EBP50 PDZ1+2 interactions. After capturing His-tagged Shank2 PDZ, the NTA chips were perfused with a fixed concentration of the C-terminus of CFTR (300 nm) and increasing concentrations of the GST-tagged EBP50 PDZ1+2. EBP50 PDZ1+2 dose-dependently decreased the CFTR-Shank2 PDZ association with an IC 50 value of 91 nm (n=3). Ka: association constant, Kd: dissociation constant, KD: overall dissociation constant at equilibrium status (Kd/Ka)

23 2. Regulation of CFTR Cl - Channel Activity by CFTR-EBP50 and CFTR- Shank2 Competitions The above results imply that Shank2 and EBP50 compete for binding on CFTR. This competitive balance may affect CFTR ion transporting activities at the apical membrane of intestine, pancreas, and kidney epithelia where Shank2 and EBP50 are abundantly expressed 6,8. Therefore, I determined the effects of the competition between EBP50 and Shank2 PDZ binding on the CFTR Cl - channel activity using inside-out configurations (FIG. 2). Following patch excision from CFTR-transfected CHO cells, patch membranes were washed with a high bath flow for 5 min to eliminate endogenous activation. When CFTR was activated by the addition of the catalytic subunit of PKA and ATP to the bath solution, an ion channel activity with a single channel conductance of 7.1 ± 0.3 ps and a linear I-V relationship was evoked, which was absent in mock-transfected cells. It has been shown that scaffolds with multiple PDZ domains, such as EBP50 and CAP70/PDZK1, can activate CFTR Cl - channel activity independent of camp signals by altering protein conformation including multimerization 15,16. In agreement with these reports, treatment with EBP50 (100 nm) induced a 2.8-fold increase in the open probability (Po) of CFTR. Importantly, addition of Shank2 PDZ (300 nm) inhibited the EBP50-induced increase in CFTR Po by 75 ± 8% (FIG. 2, a and c). The effect of Shank2 PDZ versus EBP50 competition was then examined in reverse order. Shank2 PDZ treatment alone did not evoke significant changes in CFTR Po at basal levels however it greatly inhibited the CFTR activation mediated by EBP50. Addition of EBP50 induced only a minor increase in CFTR Po after Shank2 pre-treatment (FIG. 2, b and d). When performing the

24 above experiments using EBP50 PDZ1+2 which does not bind AKAPs due to the lack of the ERM domain, I obtained results similar to those using the entire EBP50 protein (FIG. 2, c and d)

25 a Inside-out patch Washout Add PKA Basal Add EBP50 Add Shank PDZ Inside-out configurations min O C O C +60 mv Basal (P o =0.098) EBP50 (P o =0.415) PDZ2 PDZ1 ERM EBP50 + Shank2 PDZ O C EBP50 + Shank2 PDZ (P o =0.195) 0.5 pa 5 s b Inside-out patch Washout Add PKA Add Shank PDZ Basal Add EBP50 Inside-out configurations O C O C min +60 mv Basal (P o =0.114) Shank2 PDZ (P o =0.141) Shank2 PDZ + PDZ2 PDZ1 ERM EBP50 O C Shank2 PDZ + EBP50 (P o =0.198) 0.5 pa 5 s c P O ( % o v e r b a s a l) EBP50 (whole) Shank2 PDZ EBP50 PDZ1+2 Shank2 PDZ d P O ( % o v e r b a s a l) EBP50 (whole) Shank2 PDZ EBP50 PDZ1+2 Shank2 PDZ

26 FIGURE 2. Regulation of CFTR single channel activity by a competition between CFTR-EBP50 and CFTR-Shank2 PDZ binding CFTR Cl - channel activity was measured in CFTR-transfected CHO cells with inside-out configurations. Current records at +60 mv were analyzed to estimate the open probability (P o ) (a) After a 5-min wash of patch membranes, CFTR was activated by addition of PKA. Solutions containing EBP50 (100 nm) with and without Shank2 PDZ (300 nm) were perfused to the bath chamber at 5-min intervals. P o was calculated from the recording of the last 1 min of each 5 min interval. Similar experiments were performed with EBP50 PDZ1+2, which lacks the AKAP/PKA-binding ERM domain, instead of EBP50 (whole). A summary of five experiments with EBP50 and three experiments with EBP50 PDZ1+2 is presented in panel c. (b) The effect of Shank2 PDZ and EBP50 binding on CFTR activity was examined in reverse order of panel a, with Shank2 PDZ treatment preceding the EBP50 treatment. A summary of five experiments with EBP50 and three experiments with EBP50 PDZ1+2 is presented in panel d

27 3. Shank2 Associates with PDE4D An important clue was obtained in an experiment with the nonselective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX) (FIG. 3, a-d). Stimulation of CFTR-expressing CHO cells with 3 µm forskolin induced a CFTR Cl - current that is dependent on the presence of Cl - in bath and pipette solutions, is inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate, and has linear I-V relationships in whole-cell configurations. However, when Shank2 was coexpressed, the same amount of forskolin produced very small, or no CFTR currents. Interestingly, additional treatment with IBMX evoked a robust CFTR current in Shank2-expressing cells, although its peak amplitude was still 56 ± 7% that of mock-transfected cells (FIG. 3, a and b). These results suggest a functional association between the PDE activity and the Shank2-mediated inhibition of CFTR. Next I identified the PDE subtypes involved, and determined whether Shank2 directly interacts with PDEs. Of the 11 PDE families, PDE3 and PDE4 are strong candidates for Shank2-association as they are sensitive to IBMX and are known to be expressed in epithelial cells 17. The PDE3 family consists of two genes, 3A and 3B, and the PDE4 family of four, 4A - 4D. RT-PCR results using primers specific to the common regions of mouse and human PDEs revealed that several PDE3 and PDE4 genes, including PDE3A, 4A, and 4D, are expressed in mouse fibroblast NIH 3T3 cells and human colonic T84 cells (FIG. 3e). As both PDE3 and PDE4 are expressed, the patch clamp experiment shown in FIG. 3b was repeated after pretreatment of the cells with the PDE3-specific inhibitor, trequinsin and the PDE4-specific inhibitor, rolipram (FIG. 3, c and d). PDE3 inhibition did not alter the effects of

28 Shank2 (FIG. 3c). Conversely, when PDE4 was inhibited by rolipram, 3 µm forskolin consistently induced a robust CFTR current in Shank2-expressing cells (FIG. 3d, n=5), strongly suggesting an association between Shank2 and PDE4 isoforms. Recently, two reports suggested that PDE4D plays a major role in forming a camp diffusion barrier at the apical regions of epithelial cells 18,19. Consistent with this notion, expression of PDE4D proteins was observed in the immunoblots of NIH 3T3, CHO, and T84 cells (FIG. 3f) in which the inhibitory effects of Shank2 on camp/pka signals were demonstrated in this and previous studies 8,9. To probe for a physical association between Shank2 and PDE4D, coimmunoprecipiations(co-ips) were performed. Human and rat PDE4D loci have multiple transcriptional units that code for at least 9 splice variants, PDE4D1 to PDE4D9 14. Among them, PDE4D5 has been shown to be expressed in epithelial cells and to mediate camp hydrolysis at the apical microdomain 18. Thus, Shank2 and the PDE4D splice variant, PDE4D5, were overexpressed in CHO cells followed by IP with antibodies against Shank2 and PDE4D5 (FIG. 3g). Shank2 proteins were observed in immunoprecipitates with PDE4D5-specific antibodies. In a converse experiment, PDE4D5 could be detected in Shank2 immunoprecipitates. These co-ips were specific as they could only be detected in cells expressing both Shank2 and PDE4D5 (FIG. 3g)

29 ana 2 min Mock b Shank na Forskolin 3 µm -1.0 Forskolin 3 µm -1.0 Forskolin 10 µm IBMX 100 µm c na Shank2 PDE3 inhibition d Shank2 PDE4 inhibition Trequinsin 1 µm Rolipram 10 µm na Forskolin 3 µm Forskolin 10 µm IBMX 100 µm -1.0 Forskolin 3 µm e bp bp RT-PCR PDE isoforms M 3A 3B 4A 4B 4C 4D NIH 3T3 T84 f 3T3 CHO T84 PDE4D immunoblot rpde4d splice variants D1 D2 D3 D4 D5 PDE4D1/2/6 (short forms) PDE4D3/8/9 PDE4D5/7 IB: PAN-PDE4D KDa

30 g Input Transfection Mock Sh2 Sh2/4D5 Transfection Mock Sh2 Sh2/4D KDa IB: Shank2 KDa IB: PDE4D5 Co-IP IP: PDE4D5, IB: Shank IP: Shank2, IB: PDE4D5 Figure 3. Shank2 associates with PDE4D (a-d) CFTR Cl - channel activity at whole cell configurations was measured in CFTR-expressing CHO cells with or without Shank2 co-transfection. Treatment with forskolin (3 µm) evoked a large Cl - current in mock-transfected cells (a), but not in Shank2-transfected cells (b). CFTR currents in Shank2-transfected cells were restored by treatment with the nonspecific PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) (b), or the PDE4-specific inhibitor, rolipram (d), but not with the PDE3-specific inhibitor, trequinsin (c). (e) Detection of PDE3 and PDE4 isoforms in NIH 3T3 cells (mouse fibroblast) and T84 cells (human colonic epithelia) by RT-PCR. Primer sequences specific to PDE3 and PDE4 isoforms were selected from the regions common to both mouse and human PDEs. (f) Expression of PDE4D proteins in NIH 3T3, CHO (Chinese hamster ovary), and T84 cells was analyzed by immunoblotting using anti-pan PDE4D ab Several recombinant PDE4D splice variants are shown for comparison. (g) Shank2 and the PDE4D splice variant PDE4D5 were overexpressed in CHO cells (upper panels) and subsequently immunoprecipitated from cell extracts using anti-pde4d5 (lower left panel) and anti-shank2 (lower right panel) antibodies

31 In order to identify its PDE4D-binding domain, several truncated Shank2 constructs were generated and used for pull-down assays. Shank2 has multiple sites for possible protein-protein interactions, including a PDZ domain, a long proline-rich (PR) region, and a SAM (sterile alpha motif) domain 20. The PR region contains five proline-rich clusters (PRCs), including a cortactinbinding domain (ppi). In initial pull-down assays using the lysates from the PDE4D5-trasfected CHO cells, PDE4D5 showed an interaction with the PR region of Shank2 (FIG. 4b, left). The PR region was then further dissected into two parts, PR1 and PR2, and the PDE4D binding site was mapped to the PR2 region (FIG. 4b, right) where three PRCs and a ppi are clustered (FIG. 4a). Next, all PDE4D splice variants were coimmunoprecipitated with Shank2 in order to identify the PDE4D variants that interact with Shank2 and to identify the putative Shank2-binding site in PDE4D. COS-7 cells were transfected with plasmids encoding Shank2 and the nine PDE4D splice variants, PDE4D1 to PDE4D9, followed by immunoprecipitation using the PAN-PDE4D antibody, M3S1. All PDE4D long forms (D3, D4, D5, D7, D8, and D9) were found to interact with Shank2, whereas the short forms, D2 and D6, do not (FIG. 4d). A weak interaction was detected in the case of PDE4D1. The PDE4D splice variants are distinguished by the presence or absence of two conserved N- terminal domains called upstream conserved regions 1 and 2 (UCR1 and UCR2, FIG. 4c). The above results imply that UCR1 and the N-terminal part of UCR2 mediate Shank2 binding

32 a PRCs Shank2 ppl b Pull-Down pgex PDZ PR SAM Pull-Down pgex PR1 PR2 PR PDZ Proline-rich (PR) PR1 PR2 SAM 105 KDa 75 KDa GST-fusion constructs Transfection: rpde4d5, IB: PDE4D5 c N-term PDE4D splice variants Putative Shank2 binding site UCR1 UCR2 Catalytic Domain PDE4D1 PDE4D2 PDE4D3 PDE4D4 PDE4D5 PDE4D6 PDE4D7 PDE4D8 PDE4D9 C-term d PDE4D D1 D2 D3 D4 D5 D6 D7 D8 D9 Shank Input IP: Pan-PDE4D Transfection IB: Shank2 IB: PAN-PDE4D IB: Shank2 IB: PAN-PDE4D FIGURE 4. Domains responsible for the association between Shank2 and PDE4D (a,b) to identify the PDE4D-binding domain of Shank2, pulldown assays were performed using GST fusion proteins containing different domains of Shank2 and PDE4D expressed in CHO cells. Domain structures of the GST fusion proteins are illustrated in a, and pulldown results are shown in b. Lysates from rpde4d5-transfected CHO cells were pulled down with GST-tagged Shank2 fragments and blotted with anti-pde4d5 antibody. (c,d) Immunoprecipitation of Shank2 with the nine PDE4D splice variants. COS-7 cells were transfected with plasmids encoding Shank2 and the PDE4D splice variants, PDE4D1 to PDE4D9 (D1 D9). After 3 days of culture, cells were harvested, and the resulting detergent extracts were subjected to IP with the pan- PDE4D antibody M3S1. The domain structures of PDE4D splice variants are illustrated in c, and IP results are presented in d. PRCs, proline-rich clusters; SAM, sterile motif; IB, immunoblot

33 4. Shank2 and PDE4D associate in vivo The association between Shank2 and PDE4D was then examined in cells that natively express both proteins in order to explore its physiological role. Initially, I investigated the expression of Shank2 and PDE4D in rat colonic mucosa. Shank2 and PDE4D were highly co-localized at the apical region of colonic crypt cells where CFTR plays a major role in fluid and ion secretion (FIG. 5a). The Shank2-PDE4D interaction was also verified by immunoprecipitation of the endogenous proteins prepared from T84 human colonic epithelial cells. It is well known that PDE4 can be activated by PKAinduced phosphorylation at UCR1 22. Interestingly, stimulation of the camp/pka pathway using the adenylyl cyclase activator, forskolin, increased the physical association between Shank2 and PDE4D in T84 cells (FIG. 5b). Finally, the Shank2-PDE4D interaction was examined in mouse brain tissues where multiple PDE isoforms as well as Shank2 are expressed at high levels. In immunoprecipitates using PAN-PDE4D antibodies, multiple Shank2 immunoreactive bands were detected in addition to the typical 170 kda Shank2 (FIG. 5c) due to the presence of distinct Shank2 splice variants in brain tissue. Mouse brain tissue was then immunoprecipitated with anti-shank2 antibodies and the resulting IP pellets were subjected to PDE activity assays. The PDE activity coimmunoprecipitating with Shank2 was inhibited by the PDE4 selective inhibitor, rolipram (FIG. 5d). In addition, the co-immunprecipitation of PDE was ablated in IPs using tissue from PDE4D knock-out mice (FIG. 5e) indicating that Shank2 specifically interacts with PDE4D in mouse brain tissue

34 a Colon PDE4D Shank2 Merge b T84 cells C Brain 160 KDa Input IB: Shank2 105 KDa Input IB: Shank2 160 KDa 105 KDa IB: PAN-PDE4D IP IB: Shank2 IP: PAN-PDE4D Ab Forskolin (10 µm) KDa IP IB: PAN-PDE4D IB: Shank2 IP: PAN-PDE4D Ab KDa d PDE activity (pmol/min/mg extract) Rolipram +Rolipram IgG anti-shank2 IP from mouse brain (WT) e PDE activity (pmol/min/mg extract) PDE4D-WT PDE4D-KO IgG anti-shank2 IP from mouse brain

35 FIGURE 5. Shank2 and PDE4D associate in vivo (a) immunolocalization of Shank2 and PDE4D in rat colon was detected using polyclonal anti-shank2 antibody 1136 and monoclonal pan-pde4d antibody M3S1. Shank2 and PDE4D were co-localized in the apical region of colonic crypt cells. (b) co-ip of Shank2 and PDE4D from extracts of human colonic T84 cells. Forskolin treatment (10 µm for 30 min) enhanced the association between Shank2 and PDE4D. (c) detection of Shank2 proteins in PDE4D immunoprecipitates from mouse brain. (d,e), measurement of PDE activity in Shank2 immunoprecipitates from mouse brain. In d, PDE activity in Shank2 IPs from wild type mice was determined in the presence or absence of the PDE4 specific inhibitor rolipram (10 µm). In e, PDE activity in immunoprecipitates from PDE4D wild type mice (PDE4D-WT) is compared with immunoprecipitates from PDE4D knock-out mice (PDE4D-KO). Ab, antibody; IB, immunoblot

36 IV. DISCUSSION The regulatory mechanisms resulting from the association of CFTR with EBP50 and Shank2 are summarized in Figure 6. It is known that EBP50 can activate CFTR through two independent mechanisms. First, EBP50 can recruit ezrin, a PKA anchoring protein (AKAP), and hence facilitates the camp/pka-mediated phosphorylation of CFTR 6. Second, EBP50 can activate CFTR by conformational changes, possibly by forming a CFTR dimer 15. The results of this study revealed that Shank2 can inhibit both mechanisms by competing with EBP50 at the C-terminus of CFTR. The finding that Shank2 inhibited the EBP50-induced increase of CFTR P o in inside-out patches (Fig. 2) demonstrated that Shank2 can antagonize the EBP50 function by breaching the EBP50-induced active conformation of CFTR independent of camp signals. Even more interesting, this study shows that Shank2 can ablate camp signaling in the vicinity of the CFTR by tethering PDE4D into this macromolecular complex. It is well known that camp signals are compartmentalized in many cell types, and that localized camp signals play a critical role in various physiological processes 24. For example, localized camp accumulation in a region overlapping with the Z band and the T tubules in cardiomyocytes is essential for the regulation of cardiac myocyte contraction by β-adrenergic receptors 22. Several recent reports underscored the importance of PDE4D as a camp diffusion barrier formed at the apical membrane of epithelia for the proper functioning of CFTR 18, 19. However, the molecular mechanisms for PDE accumulation in apical regions remained obscure. A thorough molecular

37 characterization in this study revealed that the apical adaptor, Shank2, recruits PDE4D by a direct interaction between the proline-rich region of Shank2 and the UCR1/2 region of PDE4D. Recently, Shank2E, a splice variant of Shank2 with additional ankyrin repeats and an SH3 domain, was identified in epithelial cells 24. Therefore, I repeated the experiments in Figure 3 with Shank2E. The results were nearly identical to those obtained using Shank2 suggesting that domains within the structure of Shank2 are important for regulating CFTR function. The physiological significance of the Shank2-PDE4D association is not confined to epithelial cells but can be extended to other organs. For example, Shank2 is widely expressed in many regions of the brain including cortex, hippocampus, and cerebellum. Shank proteins are the key organizer of PSD and interact with Homer 20, 25, which is required for efficient signaling of metabotropic glutamate receptors (mglurs) that play a critical role in learning and memory. Effector systems of mglurs are not only associated with Ca 2+ signaling but also with camp signals, and it is believed that camp signals play a role in the functional specificity of each mglur isoform 27,28. Therefore, Shank2-associated PDE4D may play a critical role in the regulation of PSD in neuronal cells. Recent studies with knock-out animals of NHEFR family PDZ proteins (EBP50, E3KARP, and PDZK1) have shown that the functions of these adaptors are more complex than previously appreciated and appear to be tissue-specific 5. Because PDZ domains have well-defined binding sites, they are promising targets for drug discovery 21. However, as the present study demonstrates, much

38 is yet to learn about the function of each PDZ-based adaptor before drugs targeting PDZ interactions can become a reality 26. For example, as the PDZ structures of EBP50 and Shank2 are very similar, small molecules or peptides targeting EBP50 may have untoward effects in vivo by disrupting Shank2 complexes as well. In conclusion, the present study demonstrates the functional diversity of PDZ-mediated protein-protein interactions and illustrates that opposite signals can be delivered to the same PDZ-binding motif of a given membrane protein by different adaptors. A competitive balance between the signal-conferring and signal-stopping PDZ interactions would be critical in the regulation of many membrane transporters and receptors, as demonstrated here for CFTR

39 Cl -, HCO 3 - CFTR NBD1 R NBD2 EBP50/NHERF1 + - PDZ-binding motif Shank2 ERM Ezrin R R C C PDZ2 PDZ1 PKA SAM camp PDZ PDE4D Proline-rich FIGURE 6. A model for the regulation of CFTR through interaction with EBP50 and Shank2 Through their respective PDZ domains, Shank2 and EBP50 compete for binding of the CFTR C terminus. Binding of EBP50 activates CFTR through a conformational change. In addition, EBP50 facilitates the PKA-mediated phosphorylation and activation of CFTR by bringing the AKAP ezrin and PKA into the protein complex. Shank2 inhibits CFTR activity by breaching the CFTR-EBP50 association and by bringing PDE4D, which precludes camp/pka signaling, closer to CFTR. SAM, sterile motif; R of CFTR, R-domain; R and C of PKA, regulatory and catalytic subunits

40 V. CONCLUSION The present study shows the kinetic property and physiological significance of the interactions between CFTR and the PDZ-based scaffolds, EBP50 and Shank2. Using molecular, biophysical approaches, we conclude that: 1. 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. 2. CFTR Cl - channel activity was dynamically regulated by the competition of Shank2 and EBP50 binding. 3. Shank2 associates with PDE4D in vitro and vivo. UCR1 or UCR2 domain of PDE4D and latter Proline-Rich domain of Shank2 participate in interaction of PDE4D and Shank2, respectively. 4. In contrast to the PKA/AKAP-recruitment by EBP50, Shank2 was found to tether PDE4D to the CFTR complex, thus, attenuating camp/pka signals. These results strongly suggest that balanced interactions between the membrane transporters and multiple PDZ-based adaptors play a role in the homeostatic regulation of epithelial transport, and possibly the membrane transport in other tissues

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