A Novel PDZ Protein Regulates the Activity of Guanylyl Cyclase C, the Heat-stable Enterotoxin Receptor
2002; Elsevier BV; Volume: 277; Issue: 25 Linguagem: Inglês
10.1074/jbc.m202434200
ISSN1083-351X
AutoresRobert O. Scott, William R. Thelin, Sharon L. Milgram,
Tópico(s)Escherichia coli research studies
ResumoSecretory diarrhea is the leading cause of infectious diarrhea in humans. Secretory diarrhea may be caused by binding of heat-stable enterotoxins to the intestinal receptor guanylyl cyclase C (GCC). Activation of GCC catalyzes the formation of cGMP, initiating a signaling cascade that opens the cystic fibrosis transmembrane conductance regulator chloride channel at the apical cell surface. To identify proteins that regulate the trafficking or function of GCC, we used the unique COOH terminus of GCC as the "bait" to screen a human intestinal yeast two-hybrid library. We identified a novel protein, IKEPP (intestinal andkidney-enriched PDZprotein) that associates with the COOH terminus of GCC in biochemical assays and by co-immunoprecipitation. IKEPP is expressed in the intestinal epithelium, where it is preferentially accumulated at the apical surface. The GCC-IKEPP interaction is not required for the efficient targeting of GCC to the apical cell surface. Rather, the association with IKEPP significantly inhibits heat-stable enterotoxin-mediated activation of GCC. Our findings are the first to identify a regulatory protein that associates with GCC to modulate the catalytic activity of the enzyme and provides new insights in mechanisms that regulate GCC activity in response to bacterial toxin. Secretory diarrhea is the leading cause of infectious diarrhea in humans. Secretory diarrhea may be caused by binding of heat-stable enterotoxins to the intestinal receptor guanylyl cyclase C (GCC). Activation of GCC catalyzes the formation of cGMP, initiating a signaling cascade that opens the cystic fibrosis transmembrane conductance regulator chloride channel at the apical cell surface. To identify proteins that regulate the trafficking or function of GCC, we used the unique COOH terminus of GCC as the "bait" to screen a human intestinal yeast two-hybrid library. We identified a novel protein, IKEPP (intestinal andkidney-enriched PDZprotein) that associates with the COOH terminus of GCC in biochemical assays and by co-immunoprecipitation. IKEPP is expressed in the intestinal epithelium, where it is preferentially accumulated at the apical surface. The GCC-IKEPP interaction is not required for the efficient targeting of GCC to the apical cell surface. Rather, the association with IKEPP significantly inhibits heat-stable enterotoxin-mediated activation of GCC. Our findings are the first to identify a regulatory protein that associates with GCC to modulate the catalytic activity of the enzyme and provides new insights in mechanisms that regulate GCC activity in response to bacterial toxin. heat-stable enterotoxin COOH-terminal extension peptide postsynaptic density-95, disks large, zonula occludens-1 binding domain hemagglutinin Madin-Darby canine kidney tetramethylrhodamine isothiocyanate Guanylyl cyclase C (GCC) is the receptor for heat-stable enterotoxins (STa)1 secreted by Escherichia coli and other enteric bacteria. STa binding to GCC increases intracellular cGMP and initiates a signaling cascade, leading to the phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) at the apical surface of gastrointestinal epithelial cells. Phosphorylation of CFTR opens the channel, resulting in the net efflux of ions and water into the intestinal lumen. The endogenous ligands for GCC include guanylin, uroguanylin, and lymphoguanylin, which are thought to regulate ion transport in epithelial tissues (1Currie M.G. Fok K.F. Kato J. Moore R.J. Hamra F.K. Duffin K.L. Smith C.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 947-951Crossref PubMed Scopus (510) Google Scholar, 2Hamra F.K. Forte L.R. Eber S.L. Pidhorodeckyj N.V. Krause W.J. Freeman R.H. Chin D.T. Tompkins J.A. Fok K.F. Smith C.E. et al.Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10464-10468Crossref PubMed Scopus (325) Google Scholar, 3Forte L.R. Eber S.L. Fan X. London R.M. Wang Y. Rowland L.M. Chin D.T. Freeman R.H. Krause W.J. Endocrinol. 1999; 140: 1800-1806Crossref PubMed Scopus (53) Google Scholar). GCC is a member of a family of transmembrane proteins that includes receptors for natriuretic peptides and egg-activating peptides as well as several orphan receptors (4Wedel B. Garbers D.L. Annu. Rev. Physiol. 2001; 63: 215-233Crossref PubMed Scopus (111) Google Scholar). All receptor GCs with a single transmembrane domain share a common topology. There is an NH2-terminal extracellular ligand-binding domain and a large cytosolic domain composed of a kinase homology domain and a catalytic domain. Following the catalytic domain, GCC contains an extended COOH terminus of 63 amino acids (COOH-terminal extensionpeptide (CTEP)) that is not found in the natriuretic peptide receptors (5Schulz S. Green C.K. Yuen P.S. Garbers D.L. Cell. 1990; 63: 941-948Abstract Full Text PDF PubMed Scopus (521) Google Scholar). The CTEP is well conserved and contains a consensus protein kinase C phosphorylation site that potentiates cGMP-mediated signaling by phorbol esters (6Wada A. Hasegawa M. Matsumoto K. Niidome T. Kawano Y. Hidaka Y. Padilla P.I. Kurazono H. Shimonishi Y. Hirayama T. FEBS Lett. 1996; 384: 75-77Crossref PubMed Scopus (34) Google Scholar). GCC proteins lacking the 63-amino acid CTEP lose the ability to respond to STa (6Wada A. Hasegawa M. Matsumoto K. Niidome T. Kawano Y. Hidaka Y. Padilla P.I. Kurazono H. Shimonishi Y. Hirayama T. FEBS Lett. 1996; 384: 75-77Crossref PubMed Scopus (34) Google Scholar, 7Deshmane S.P. Parkinson S.J. Crupper S.S. Robertson D.C. Schulz S. Waldman S.A. Biochemistry. 1997; 36: 12921-12929Crossref PubMed Scopus (23) Google Scholar), suggesting that this unique sequence plays a role in GCC activation. Since GCC is the only receptor guanylyl cyclase localized predominately at the apical membrane of epithelial cells, CTEP may also play a role targeting the receptor to the apical cell surface. To determine whether the COOH terminus of GCC participates in protein-protein interactions that may regulate its targeting or function, we screened a human intestinal epithelial enriched yeast two-hybrid library using CTEP as "bait." We found that GCC associates via its COOH terminus with a novel protein containing four PDZ domains. Based on its domain organization and restricted mRNA distribution, we named this protein IKEPP (intestinal andkidney enriched PDZProtein). IKEPP is accumulated at the apical membrane of human intestinal epithelial cells and associates with GCC in a cellular context. Mutagenesis studies indicate that association with PDZ proteins is not required for efficient targeting of GCC to the apical surface. Rather, the interaction of IKEPP and GCC inhibits receptor activation by STa. Thus, GCC activity may be modulated by interaction with accessory proteins, thereby providing additional means to regulate signaling via guanylyl cyclase receptors. All cDNA inserts were generated by PCR, cloned into complementary restriction endonuclease sites of the appropriate plasmids, and verified by sequencing; specific details are available upon request. A human intestinal epithelial enriched cDNA library was generated by cloning poly(dT)-primed cDNA into the HybriZAP bacteriophage λ vector followed by amplification and in vivo mass excision to generate a two-hybrid library in pAD-GAL4 (Stratagene). The yeast binding domain (BD) plasmid pPC86BD was generated by digesting the parental vectors, pPC97 (GAL4BD and LEU2) and pPC86 (GAL4AD and TRP1) (8Chevray P.M. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5789-5793Crossref PubMed Scopus (481) Google Scholar), with ApaI andBamHI. These fragments were then ligated into the opposite backbone vector to give pPC86BD and pPC97AD. cDNA encoding full-length CTEP was amplified by PCR using pBS.GCC as template; the PCR products were inserted in frame to the corresponding sites in pPC86BD. The yeast strain AH109 was sequentially transformed with pPC86BD.CTEP and 20 μg of a human intestinal cDNA library as described (9Smith F.D. Oxford G.S. Milgram S.L. J. Bio. l Chem. 1999; 274: 19894-19900Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). A ∼2.2-kb cDNA clone encoding a novel protein was isolated twice in the screen. After sequencing and Northern blot analysis, this clone was named IKEPP (intestinal and kidney enrichedPDZ protein). To obtain upstream coding sequences we performed 5′ rapid amplification of cDNA ends using Marathon-Ready Human Kidney cDNA (CLONTECH); products were cloned into pTAdv (CLONTECH) and sequenced. Human multiple tissue northern blots and a multiple expression array blot (CLONTECH) were probed with32P-labeled random-primed cDNA probe corresponding to the IKEPP 3′-untranslated region (nucleotides 1565–2120) as described (10Trotter K.W. Fraser I.D. Scott G.K. Stutts M.J. Scott J.D. Milgram S.L. J. Cell Biol. 1999; 147: 1481-1492Crossref PubMed Scopus (83) Google Scholar). Rabbit antisera directed against the COOH terminus of human IKEPP were generated in rabbits using residues 484–505 of IKEPP coupled with keyhole limpet cyanin as immunogen. Rabbit polyclonal antisera were also generated using His-IKEPP fusion protein as immunogen. The pET.IKEPP plasmid was transformed into BL21(DE3, pLysS) Escherichia coli and grown to the appropriate cell density at 37 °C. IKEPP expression was induced by the addition of 1 mmisopropyl-1-thio-β-d-galactopyranoside for 3 h at 37 °C and purified from the insoluble fraction. To prepare cell lysates, cultured cells were washed with ice-cold phosphate-buffered saline (50 mm NaPO4, 150 mm NaCl, pH 7.4) and isolated by scraping in ice-cold homogenization buffer containing 20 mm Hepes, pH 7.4, 150 mm NaCl, 2 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin. The homogenates were centrifuged at 100,000 ×g for 1 h to generate soluble and particulate fractions. Protein concentrations were determined using the BCA protein assay kit (Pierce); samples were fractionated by SDS-PAGE and transferred to Immobilon-P (Millipore Corp.). Western blots were performed using rabbit anti-IKEPP IgG (NC368 or NC369; 1:2000) and visualized using ECL. In vitro binding assays and co-immunoprecipitations were performed as described (11Mohler P.J. Kreda S.M. Boucher R.C. Sudol M. Stutts M.J. Milgram S.L. J. Cell Biol. 1999; 147: 879-890Crossref PubMed Scopus (166) Google Scholar). For immunoprecipitation of overexpressed HA-GCC and IKEPP, COS7 cells were transfected with cDNAs encoding IKEPP and HA-GCC or HA-GCCΔ4 with FuGENE6 (Roche Molecular Biochemicals). After 48 h, the cells were lysed in TBS (100 mm Tris-HCl, 150 mmNaCl, pH 7.5), 1% Triton X-100, and protease inhibitors. Mouse anti-HA or purified normal mouse IgG (2 μg) was added to the cell lysate and incubated overnight at 4 °C. Immune complexes were collected on protein G-agarose and washed extensively in TBS buffer plus 0.1% Triton X-100. Bound proteins were resolved by SDS-PAGE and analyzed by Western blotting with HA or IKEPP antisera. Stable MCDK type II cell lines expressing HA-GCC or HA-GCCΔ4 were generated as described (10Trotter K.W. Fraser I.D. Scott G.K. Stutts M.J. Scott J.D. Milgram S.L. J. Cell Biol. 1999; 147: 1481-1492Crossref PubMed Scopus (83) Google Scholar). MDCK or Caco2 cells were grown on Transwell filters (Costar) until confluent monolayers were observed, and transepithelial resistances, with filter subtraction, were greater than 1000 ohms·cm2 or 400 ohms·cm2, respectively. Immunofluorescent staining was performed as described (10Trotter K.W. Fraser I.D. Scott G.K. Stutts M.J. Scott J.D. Milgram S.L. J. Cell Biol. 1999; 147: 1481-1492Crossref PubMed Scopus (83) Google Scholar, 11Mohler P.J. Kreda S.M. Boucher R.C. Sudol M. Stutts M.J. Milgram S.L. J. Cell Biol. 1999; 147: 879-890Crossref PubMed Scopus (166) Google Scholar). The localization of IKEPP was also studied in sections of formalin-fixed human colon and small intestine. Sections were prepared as described previously (12Pucilowska J.B. McNaughton K.K. Mohapatra N.K. Hoyt E.C. Zimmermann E.M. Sartor R.B. Lund P.K. Am. J. Physioll. 2000; 279: G1307-G1322Crossref PubMed Google Scholar), stained with rabbit anti-IKEPP IgG (NC369; diluted 1:1500), and processed using the Vectastain Elite ABC kit (Vector Laboratories, Inc.); sections were counterstained with methyl green to label nuclei. COS7 cells, plated on six-well culture dishes at a density of 4 × 105 24 h prior to transfection, were incubated in FuGENE 6 as described in the instruction manual. After 48 h, the culture medium was removed, the cells were incubated in serum-free Dulbecco's modified Eagle's medium/F-12 containing 100 μm isobutylmethylxanthine for 15 min, and 25 units/ml STa (Sigma) was added to each well for 20 min. The cells were washed twice in ice-cold phosphate-buffered saline, lysed in 0.1 m HCl for 20 min, and collected by centrifugation at 4 °C. For dose-response curves, cells were handled as described except that 2 × 105 cells were seeded in 12-well culture dishes 24 h prior to transfection. Following transfection, STa was added to the cells at various concentrations for 30 min in the presence of 100 μm isobutylmethylxanthine. Cells were harvested, and cGMP production was measured in both the cell lysate and culture medium using a Correlate-EIA Direct Cyclic GMP Enzyme Immunoassay Kit (Assay Designs, Inc.). In an attempt to isolate GCC-interacting proteins, we used the yeast two-hybrid system to identify proteins that interact with the CTEP of GCC. Screening of a human epithelial enriched intestinal cDNA library yielded several potential interactors that were His+, and we further analyzed two clones that exhibited robust β-galactosidase activity. The specificity of the interaction in yeast was verified by transforming the activation domain plasmid along with the original bait, an empty bait vector, or a plasmid encoding an unrelated bait (data not shown). Sequence analysis revealed that the cDNA inserts were ∼2.4 kb and contained identical cDNA sequence with an open reading frame of 1503 nucleotides. A protein pattern search using Pfam indicated that the open reading frame encoded a protein containing four PDZ domains. The gene was mapped to a region of chromosome 11q23 when searched against the human genome draft data base. The full-length cDNA with an open reading frame of 1518 nucleotides was predicted from genomic DNA and confirmed by 5′ rapid amplification of cDNA ends using human kidney cDNA as template. The open reading frame predicts a protein of 505 amino acids with a theoretical molecular mass of 54.2 kilodaltons and a pI of 5.46. On Northern blots, we detected ∼2.3- and 2.5-kb messages in human kidney (Fig. 1 A), although prolonged exposures of the blots revealed that the mRNAs were also expressed in the small intestine and colon. Since GCC mRNA is abundantly expressed in the intestine (5Schulz S. Green C.K. Yuen P.S. Garbers D.L. Cell. 1990; 63: 941-948Abstract Full Text PDF PubMed Scopus (521) Google Scholar), we also probed a human expression array containing poly(A)+ RNA prepared from multiple gastrointestinal tissues. We found that mRNA was easily detected in the kidney and along the entire gastrointestinal tract, from the duodenum to the colon (Fig. 1 B). The mRNA was not detected in any other human tissue including brain, heart, skeletal muscle, or cells of hematopoietic origin (data not shown). Based on the relatively restricted distribution of the mRNA and the domain structure of the predicted protein, we named this novel protein IKEPP. A BLAST search of the nonredundant GenBankTM data base with the IKEPP protein sequence revealed that IKEPP is most closely related to PDZK1 (PDZ domain containing protein-1; also called CAP70), a protein with four PDZ domains (13Kocher O. Comella N. Gilchrist A. Pal R. Tognazzi K. Brown L.F. Knoll J.H. Lab. Invest. 1999; 79: 1161-1170PubMed Google Scholar, 14Wang S. Yue H. Derin R.B. Guggino W.B. Li M. Cell. 2000; 103: 169-179Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). IKEPP and PDZK1 are closely related to two other human epithelial PDZ proteins, EBP50 (ezrin-radixin-moesin-bindingphosphoprotein-50; also called NHERF1) and E3KARP (NHE3kinase Aregulatory protein; also called NHERF2), which each contain two PDZ domains (Fig. 1 C) followed by a COOH-terminal domain that associates with the NH2 terminus of ezrin, radixin, and moesin to link these proteins to the actin cytoskeleton (15Reczek D. Berryman M. Bretscher A. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (518) Google Scholar). An analysis of the sequence identity between the individual IKEPP PDZ domains and the PDZ domains of EBP50, E3KARP, and PDZK1 indicates that PDZ1 and PDZ4 of IKEPP are most unique, whereas PDZ2 and PDZ3 of IKEPP share between 30 and 50% identity with the PDZ domains of these related proteins (Fig. 1 D). Furthermore, IKEPP is probably the human orthologue of the mouse type IIa sodium/inorganic phosphate cotransporter-associated protein (Na/Pi-Cap2), since both proteins contain four tandem PDZ domains, share 77% sequence identity, and are expressed in the kidney and intestine (16Gisler S.M. Staglijar I. Traebert M. Bacic D. Biber J. Murer H. J. Biol. Chem. 2001; 276: 9206-9213Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). PDZ domains are composed of six β sheets (βA–βF), capped by two α helices (αA and αB), which form a peptide-binding groove that interacts with, at least, the last four C-terminal amino acids of interacting proteins (17Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (976) Google Scholar). In general, PDZ domains recognize peptide sequences that contain a hydrophobic residue at the extreme COOH terminus through a conserved carboxylate-binding pocket most often formed by the sequence Arg/Lys-X-X-X-Φ-Gly-Phe (where Φ represents a hydrophobic amino acid) (18Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1049) Google Scholar). The carboxylate loop of PDZ domains in IKEPP, PDZK1, EBP50, and E3KARP contain the general consensus, Arg/Lys-X-X-Tyr/Phe-Gly-Phe, with the exception of IKEPP PDZ4, which possesses a Pro residue rather than the Arg/Lys (Fig. 1 D). In the carboxylate-binding pocket, the Arg/Lys residue is responsible for ordering a water molecule that interacts with the terminal carboxylate of the ligand (17Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (976) Google Scholar). Therefore, the C-terminal residue(s) of proteins that associate with IKEPP PDZ4 will probably differ from the ligands recognized by PDZK1, EBP50, E3KARP, and IKEPP PDZ domains 1–3. The −2-position of the preferred peptide ligand is used to categorize the PDZ domains as class I (−2 Ser/Thr), class II (−2 hydrophobic), and a lesser defined class III, which deviate from class I and II (18Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1049) Google Scholar, 19Harris B.Z. Lim W.A. J. Cell Sci. 2001; 114: 3219-3231Crossref PubMed Google Scholar, 20Songyang Z. Fanning A.S., Fu, C., Xu, J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1224) Google Scholar). The specificity of the −2 interaction is coordinated by the first residue of the second α helix of the PDZ domain (αB1) (18Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1049) Google Scholar). At the αB1 position, class I PDZ domains contain a conserved His residue (17Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (976) Google Scholar, 21Karthikeyan S. Teil L. Ladias J.A. J. Biol. Chem. 2001; 276: 19683-19686Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), whereas class II domains possess a hydrophobic residue (22Daniels D.L. Cohen A.R. Anderson J.M. Brünger A.T. Nat. Struct. Biol. 1998; 5: 317-325Crossref PubMed Scopus (161) Google Scholar). To the best of our knowledge, all of the published binding partners of PDZK1, EBP50, and E3KARP, as well as the binding partners our laboratory has identified for IKEPP, contain a Ser/Thr at the −2-position of the PDZ binding motif. Based on this structural similarity, we predict that IKEPP is a member of the superfamily of class I PDZ proteins. Sequence analysis of the individual PDZ domains of IKEPP, however, reveals that IKEPP PDZ1 and PDZ4 lack the conserved His residue characteristic of class I PDZ domains. In the αB1 position, a Tyr residue (IKEPP PDZ1) has been shown to prefer ligands containing a −2 Asp residue, whereas an Asp residue (IKEPP PDZ4) interacts with peptides with a −2 Tyr (20Songyang Z. Fanning A.S., Fu, C., Xu, J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1224) Google Scholar, 23Stricker N.L. Christopherson K.S., Yi, B.A. Schatz P.J. Raab R.W. Dawes G. Bassett D.E., Jr. Bredt D.S. Li M. Nat. Biotechnol. 1997; 15: 336-342Crossref PubMed Scopus (220) Google Scholar). To evaluate the subcellular distribution of IKEPP, we generated rabbit polyclonal antisera directed against the COOH-terminal 15 amino acids of human IKEPP or the recombinant full-length protein. These antisera were first tested by Western blot analysis using full-length human IKEPP generated by coupled in vitrotranscription/translation. Whereas preimmune sera did not detect proteins in the reticulocyte lysates, both antibodies reliably detected full-length IKEPP (Fig. 2 A). We further tested the specificity of our IKEPP antisera by Western blot analysis of EBP50, E3KARP, and PDZK1 and found that both IKEPP antisera specifically recognize recombinant IKEPP and do not cross-react with these related proteins (Fig. 2 A). Recombinant E3KARP contains fewer methionine residues than IKEPP, PDZK1, and EBP50 and was visualized with prolonged exposure to the PhosphorImager screen. We first examined the expression of IKEPP in cultured human cell lines and found that the protein was expressed in whole cell lysates of two intestinal epithelial cell lines, T84 and Caco2 (Fig. 2 B); much less protein was detected in an airway epithelial cell line (16HBE14o−) or in hEK293 cells. A significant fraction of the IKEPP protein was found in the particulate fraction of Caco2 and T84 cells (Fig. 2 C). We next examined the localization of IKEPP in Caco2 cells grown to confluence on Transwell filters and found IKEPP preferentially accumulated in the subapical compartment and at the apical membrane (Fig.3 A); similar results were obtained with colonic T84 cells (data not shown). In normal human ileum and colon, IKEPP was preferentially accumulated at the apical surface and was visualized in cells of the crypt and villus (Fig.3 B). GCC is also expressed at the apical surface of intestinal epithelial cells (24Nandi A. Bhandari R. Visweswariah S.S. J. Cell. Biochem. 1997; 66: 500-511Crossref PubMed Scopus (33) Google Scholar). Thus, the distribution of IKEPP in human intestine is consistent with the possibility that the GCC and IKEPP associate in vivo. We further characterized the interaction between GCC and IKEPP. Since GCC terminates with the amino acid sequence STYF, a type I PDZ binding motif, we tested whether the COOH-terminal four amino acids of CTEP mediated the interaction with IKEPP. We immobilized GST, GST-CTEP full-length, or GST-CTEPΔ4 fusion proteins on glutathione-agarose beads and incubated the affinity resins with radiolabeled IKEPP. We found that IKEPP bound GST-CTEP but not GST or GST-CTEPΔ4 (Fig.4 A). We obtained similar results in overlay assays (data not shown), indicating that the last four amino acid residues (SYTF) of GCC are required for the direct association with IKEPP. IKEPP has four PDZ domains that probably bind different ligands. Therefore, we determined which IKEPP PDZ domains are capable of associating with CTEP. To do this, we generated histidine-tagged fusion proteins consisting of PDZ1, PDZ2, PDZ3, or PDZ4 of IKEPP and tested which of the radiolabeled fusion proteins associated with full-length GST-CTEP immobilized on glutathione-agarose beads. We found that radiolabeled PDZ3 bound specifically to full-length GST-CTEP, but not GST or GST-CTEPΔ4. This interaction was not detected for PDZ1, PDZ2, and PDZ4 (Fig. 4 B). Since IKEPP shares homology with EBP50, E3KARP, and PDZK1, we immobilized GST-CTEP and GST-CTEPΔ4 on glutathione-agarose beads and tested whether radiolabeled PDZK1, EBP50, or E3KARP could associate with CTEP. We found that PDZK1, but not EBP50 or E3KARP, associates with GST-CTEP in pull-down assays (Fig. 4 C). To determine whether full-length IKEPP could associate with full-length GCC, we incubated GST or GST-IKEPP with whole cell lysates prepared from cells overexpressing HA-tagged GCC (HA-GCC). HA-GCC associated with GST-IKEPP but not with GST (Fig. 4 D). GCC may be tightly associated with the subapical cytoskeleton in the intestinal epithelium (25Waldman S.A. Kuno T. Kamisaki Y. Chang L.Y. Gariepy J. O'Hanley P. Schoolnik G. Murad F. Infect. Immun. 1986; 51: 320-326Crossref PubMed Google Scholar) and is not easily solubilized from cell membranes in buffers compatible with maintaining protein-protein interactions. Therefore we used an overexpression strategy to study the association of GCC and IKEPP in nonepithelial cells. COS7 cells were transiently transfected with cDNAs encoding IKEPP plus HA-GCC or IKEPP plus HA-GCCΔ4. Cell lysates were prepared in buffers containing 1% Triton X-100, which is known to remove GCC from cell membranes in COS7 cells (5Schulz S. Green C.K. Yuen P.S. Garbers D.L. Cell. 1990; 63: 941-948Abstract Full Text PDF PubMed Scopus (521) Google Scholar), and the cell lysates were incubated with control IgG or HA antibody. We found that IKEPP was not associated with control IgG but was easily detected in HA-GCC immunoprecipitates (Fig. 4 E). Moreover, IKEPP was not found in HA immunoprecipitates from cells co-expressing HA-GCCΔ4 plus IKEPP (Fig. 4 E). Thus, we conclude that GCC and IKEPP associate in cells and that the association requires an intact GCC COOH terminus. Interaction with PDZ proteins may be involved in selectively targeting proteins to apical or basolateral cell surfaces in epithelial cells (26Kim S.K. Curr. Opin. Cell Biol. 1997; 9: 853-859Crossref PubMed Scopus (106) Google Scholar, 27Brown D. Stow J.L. Physiol. Rev. 1996; 76: 245-297Crossref PubMed Scopus (179) Google Scholar, 28Straight S.W. Chen L. Karnak D. Margolis B. Mol. Biol. Cell. 2001; 12: 1329-1340Crossref PubMed Scopus (42) Google Scholar). Therefore, we tested whether the COOH-terminal STYF sequence in GCC was involved in targeting the receptor to the apical cell surface. We generated stable MDCK cell lines expressing HA-GCC or HA-GCCΔ4, lacking the STYF residues that mediate interaction with PDZ proteins. Full-length HA-GCC was targeted to the apical cell surface and was not detected at the basolateral membrane (Fig.5 A). Likewise, HA-GCCΔ4 was preferentially accumulated at the apical cell surface of polarized MDCK cells (Fig. 5 A). HA-GCC and HA-GCCΔ4 were visualized at the apical membrane in nonpermeabilized cells, further suggesting that the HA-GCC and HA-GCCΔ4 proteins were on the cell surface (data not shown). Thus, we conclude that interaction with apical membrane PDZ proteins does not play a significant role in the targeting of GCC to the apical cell surface in MDCK cells. Bakre et al. recently compared the STa-induced desenstitization of GCC in intestinal epithelial cells and in transfected fibroblasts and suggested that GCC catalytic activity might be regulated by interaction with proteins selectively expressed in epithelial cells (29Bakre M.M. Ghanekar Y. Visweswariah S.S. Eur. J. Biochem. 2000; 267: 179-187Crossref PubMed Scopus (25) Google Scholar). Therefore, we tested whether IKEPP modulated STa-mediated activation of GCC in transfected COS7 cells that do not express significant amounts of endogenous IKEPP. Treatment of COS7 cells expressing HA-GCC with 25 units/ml STa for 20 min significantly increased intracellular cGMP, whereas cGMP was undetected in mock-transfected cells (data not shown). In cells co-expressing GCC and IKEPP, 25 units/ml STa also increased intracellular cGMP above background. cGMP levels, however, were reduced by ∼1.7-fold in cells co-expressing HA-GCC and IKEPP compared with cells transfected with HA-GCC and empty vector (Fig. 5 B). In similar experiments, intracellular cGMP levels were decreased in COS7 cells expressing GCC and IKEPP by 1.5–2.5-fold compared with cells expressing HA-GCC and empty vector following incubation with 25 units/ml STa for 10–30 min (data not shown). This cannot be explained by changes in the expression of HA-GCC in the co-transfected cells, since the receptor was easily detected in membrane fractions prepared from these cells (Fig.5 B). Since the COOH terminus of GCC mediates the interaction with IKEPP, we tested whether IKEPP expression also inhibited STa-mediated activation of HA-GCCΔ4. We observed similar levels of STa-mediated cGMP in HA-GCCΔ4 cells in the absence or presence of co-expressed IKEPP (Fig. 5 B). Therefore, we conclude that IKEPP binding may inhibit the catalytic activity of GCC and that the inhibition requires a physical interaction between the receptor and IKEPP. To begin to understand the mechanism of this inhibition, we transfected COS7 cells with HA-GCC with or without IKEPP and assayed cGMP accumulation over a range of STa concentrations. Application of STa resulted in a concentration-dependent accumulation of cGMP in cells expressing HA-GCC plus vector or HA-GCC plus IKEPP (Fig.5 C). Although we found no change in theV max of the enzyme in the presence or absence of co-expressed IKEPP, we found that IKEPP s
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