Volume 137, Issue 2, Pages 607-617.e4 (August 2009) Synaptic Scaffolding Molecule Binds to and Regulates Vasoactive Intestinal Polypeptide Type-1 Receptor in Epithelial Cells  Heon Yung Gee, Young Woong Kim, Min Jae Jo, Wan Namkung, Joo Young Kim, Hyun Woo Park, Kyung Sik Kim, Hoguen Kim, Akemichi Baba, Jinhee Yang, Eunjoon Kim, Kyung Hwan Kim, Min Goo Lee  Gastroenterology  Volume 137, Issue 2, Pages 607-617.e4 (August 2009) DOI: 10.1053/j.gastro.2009.01.065 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 PDZ-based protein interaction between VPAC1 and S-SCAM in HEK 293 cells. (A) The VPAC1 receptor contains a class I PDZ-binding motif (-S/T-X-Φ) conserved in human, rat, mouse, pig, and chicken receptors. (B) To delete the C-terminal PDZ-binding motif (-EVSLV), the Glu-455 residue of mVPAC1 was substituted with a stop codon (pcDNA3.1-Flag-mVPAC1-ΔC). FLAG or YFP tags were inserted into mVPAC1after the ER signaling sequence between Ala30 and Ala31 of the N-terminal extracellular portion. (C and D) Coimmunoprecipitation (IP) between VPAC1 and S-SCAM. In panel C, HEK 293 cells were transiently transfected with pcDNA3.1-Flag-mVPAC1 and/or pcDNA3-S-SCAM plasmids. In panel D, human pancreatic and colonic tissues were used. In immunoblotting, 20 μg of protein was loaded into each lane, and IP was performed with a total of 1 mg lysate. (E) Localizations of VPAC1 and S-SCAM/MAGI-2 in human colonocytes. Arrowhead indicates that both proteins are colocalized at the junctional area near the apical end of the lateral membrane in human colonic crypt cells. DAPI, 4′,6-diamidino-2- phenylindole staining for nuclear localization. Gastroenterology 2009 137, 607-617.e4DOI: (10.1053/j.gastro.2009.01.065) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Surface expression of VPAC1 in HEK 293 and HeLa cells. (A) Surface biotinylation. HEK 293 cells expressing mVPAC1 were transfected with pcDNA3-S-SCAM or mock plasmid and exposed to VIP (1 μmol/L) for 30 minutes. Membrane proteins were biotinylated and immunoblotted with anti-FLAG antibody. (B) Surface immunocytochemistry. HeLa cells overexpressing mVPAC1 and/or S-SCAM were exposed to VIP (1 μmol/L) for 30 minutes. The receptors were labeled with anti-FLAG antibody and a secondary fluorescein isothiocyanate-conjugated antibody without plasma membrane permeabilization. (C) VPAC1 surface labeling and ELISA in HEK 293 cells. mVPAC1 surface expression was quantified as detailed in the Materials and Methods section. Values are the means ± SE of 6 experiments. **P < .01; difference from Flag-mVPAC1 only (far left-hand column). Gastroenterology 2009 137, 607-617.e4DOI: (10.1053/j.gastro.2009.01.065) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Time-lapse imaging of VPAC1 internalization. (A) HeLa cells transfected with pcDNA3.1-YFP-VPAC1 were stimulated with 1 μmol/L VIP, and sequential TIRF images were recorded. (B) Representative tracings of the VIP response in HeLa cells transfected with pcDNA3-S-SCAM or mock vector. The integrated optical density (OD) values were calculated from the TIRF image series. Original TIRF images of VPAC1 (1 μmol/L) and VPAC1 + S-SCAM (1 μmol/L) are presented in Supplementary Movies 1 and 2, respectively. (C) Summarized results of 7 experiments. The integrated OD values were measured 4 minutes after VIP application (1 μmol/L). *P < .05; difference from Flag-mVPAC1 only (far left-hand column). Gastroenterology 2009 137, 607-617.e4DOI: (10.1053/j.gastro.2009.01.065) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Effect of S-SCAM on VPAC1-stimulated cAMP accumulation. HEK 293 cells expressing mVPAC1 were transfected with pcDNA3-S-SCAM or mock plasmids and exposed to various concentrations of VIP for 30 minutes. Intracellular cAMP concentration ([cAMP]i) was measured as in the Materials and Methods section. Data represent mean ± SE for 4 independent experiments. Gastroenterology 2009 137, 607-617.e4DOI: (10.1053/j.gastro.2009.01.065) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Effect of S-SCAM on VIP-induced CFTR currents in Xenopus ooctyes. Xenopus ooctyes were injected with cRNAs of mVPAC1, CFTR, and/or S-SCAM, and VIP-induced CFTR currents were measured using a 2-electrode voltage clamp. (A) Schematic diagram showing the signaling cascades of VIP-induced CFTR Cl− currents near the oocyte membrane. (B) Summary of VIP-induced CFTR currents. Values are the means ± SE of 7 experiments recorded at a holding potential of −50 mV. (C) The current-voltage (I-V) curves were taken under basal conditions and during steady-state VIP stimulation by clamping the voltage from −50 to +50 mV at 10-mV intervals. (D and E) Representative traces of current measurements at a −50-mV holding potential from oocytes injected with mock and S-SCAM cRNAs are shown in panels D and E, respectively. **P < .01; difference from VPAC1 only. Gastroenterology 2009 137, 607-617.e4DOI: (10.1053/j.gastro.2009.01.065) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Effect of S-SCAM loss on VIP-induced CFTR currents in T84 cells. (A) T84 cells were transfected with scrambled control siRNA or S-SCAM-specific siRNA and grown on a permeable support for 3 and 12 days (left-hand column). In addition, protein samples were taken from T84 cells stably transfected with mock (T84/Mock) or antisense-S-SCAM plasmids (T84/-S-SCAM) 12 days after seeding on a permeable support (right-hand column). Equal amounts (20 μg) of protein samples were immunoblotted against S-SCAM and β-actin. (B) Summary of short-circuit current (Isc) measurements in T84/Mock and T84/Antisense-S-SCAM cells (n = 5). (C) Representative traces of Isc measurements in T84/Mock and T84/AS-S-SCAM cells. VIP application to the basolateral side evoked a lumen negative Isc that is fully inhibited by basolateral application of the Na+-K+-2Cl− cotransporter (NKCC) inhibitor bumetanide (100 μmol/L). The apical side was treated with amiloride (100 μmol/L) to block epithelial Na+ channels (ENaC). **P < .01; difference from T84/Mock. BLM, basolateral membrane; LM, luminal (apical) membrane. Gastroenterology 2009 137, 607-617.e4DOI: (10.1053/j.gastro.2009.01.065) Copyright © 2009 AGA Institute Terms and Conditions

Figure 7 Expression of VPAC1 and S-SCAM in T84 colonic epithelial cells. Localizations of VPAC1 and S-SCAM were analyzed by immunocytochemistry and domain-specific surface biotinylation. (A) Localization of natively expressed S-SCAM and VPAC1 in T84 monolayers. Fluorescent images of the vertical z-axis section were obtained after staining with anti-S-SCAM (red) and anti-VPAC1 (green; MAB5468; Millipore) using a Zeiss LSM 510 confocal microscope. (B) Domain-specific surface biotinylation of VPAC1. Note that VPAC1 is expressed only in the basolateral membrane, not in the apical membrane of the polarized T84 cells. (C) Role of S-SCAM in the cellular localization of VPAC1. T84 monolayers were transiently transfected with pcDNA3.1-Flag-mVPAC1, and vertical z-axis images were taken after staining with anti-S-SCAM (red), anti-E-cadherin (red), anti-ZO-1 (red), and anti-FLAG (green) antibodies. Images from the T84/Mock (upper panels) and T84/Antisense-S-SCAM (lower panels) cells were compared. Gastroenterology 2009 137, 607-617.e4DOI: (10.1053/j.gastro.2009.01.065) Copyright © 2009 AGA Institute Terms and Conditions

Figure 8 A model for VPAC1 regulation by S-SCAM/MAGI-2. S-SCAM binds to VPAC1 and negatively regulates VPAC1 activity to prevent overt stimulation and unnecessary activation of VPAC1. In addition, S-SCAM recruits VPAC1 to the junctional area near the apical end of the lateral membrane by formation of a VPAC1-S-SCAM-β-catenin-E-cadherin complex (see text for details). Confined localization of VPAC1 at the junctional area generates a localized cAMP signal close to the apical effectors such as CFTR. This, in turn, enables efficient electrolyte and fluid secretion in epithelial cells in response to VIP with minimal effects on the cell interior. Gastroenterology 2009 137, 607-617.e4DOI: (10.1053/j.gastro.2009.01.065) Copyright © 2009 AGA Institute Terms and Conditions