Title: Polycystin-2 Expression Is Regulated by a PC2-binding Domain in the Intracellular Portion of Fibrocystin
Abstract: Autosomal dominant (ADPKD) and autosomal recessive (ARPKD) polycystic kidney disease are caused by mutations in Pkd1/Pkd2 and Pkhd1, which encode polycystins (PCs) and fibrocystin/polyductin (FPC). Our recent study reported that a deficiency in FPC increases the severity of cystic disease in Pkd2 mutants and down-regulates PC2 in vivo, but the precise molecular mechanism of these effects is unknown (Kim, I., Fu, Y., Hui, K., Moeckel, G., Mai, W., Li, C., Liang, D., Zhao, P., Ma, J., Chen, X.-Z., George, A. L., Jr., Coffey, R. J., Feng, Z. P., and Wu, G. (2008) J. Am. Soc. Nephrol. 19, 455–468). In this study, through the use of deletion and mutagenesis strategies, we identified a PC2-binding domain in the intracellular C terminus of FPC and an FPC-binding domain in the intracellular N terminus of PC2. These binding domains provide a molecular basis for the physical interaction between PC2 and FPC. In addition, we also found that physical interaction between the binding domains of PC2 and FPC is able to prevent down-regulation of PC2 induced by loss of FPC. In vivo, we generated a mouse model of ADPKD with hypomorphic Pkd2 alleles (Pkd2nf3/nf3) and show that PC2 down-regulation is accompanied by a phenotype similar to that of Pkhd1–/– mice. These findings demonstrate a common mechanism underlying cystogenesis in ADPKD and ARPKD and provide insight into the molecular relationship between PC2 and FPC. Autosomal dominant (ADPKD) and autosomal recessive (ARPKD) polycystic kidney disease are caused by mutations in Pkd1/Pkd2 and Pkhd1, which encode polycystins (PCs) and fibrocystin/polyductin (FPC). Our recent study reported that a deficiency in FPC increases the severity of cystic disease in Pkd2 mutants and down-regulates PC2 in vivo, but the precise molecular mechanism of these effects is unknown (Kim, I., Fu, Y., Hui, K., Moeckel, G., Mai, W., Li, C., Liang, D., Zhao, P., Ma, J., Chen, X.-Z., George, A. L., Jr., Coffey, R. J., Feng, Z. P., and Wu, G. (2008) J. Am. Soc. Nephrol. 19, 455–468). In this study, through the use of deletion and mutagenesis strategies, we identified a PC2-binding domain in the intracellular C terminus of FPC and an FPC-binding domain in the intracellular N terminus of PC2. These binding domains provide a molecular basis for the physical interaction between PC2 and FPC. In addition, we also found that physical interaction between the binding domains of PC2 and FPC is able to prevent down-regulation of PC2 induced by loss of FPC. In vivo, we generated a mouse model of ADPKD with hypomorphic Pkd2 alleles (Pkd2nf3/nf3) and show that PC2 down-regulation is accompanied by a phenotype similar to that of Pkhd1–/– mice. These findings demonstrate a common mechanism underlying cystogenesis in ADPKD and ARPKD and provide insight into the molecular relationship between PC2 and FPC. Autosomal dominant polycystic kidney disease (ADPKD) 2The abbreviations used are: ADPKD, autosomal dominant polycystic kidney disease; ARPKD, autosomal recessive polycystic kidney disease; PC, polycystin; FPC, fibrocystin/polyductin; FBD, FPC-binding domain; PC2BD, PC2-binding domain; HA, hemagglutinin; co-IP, co-immunoprecipitation. 2The abbreviations used are: ADPKD, autosomal dominant polycystic kidney disease; ARPKD, autosomal recessive polycystic kidney disease; PC, polycystin; FPC, fibrocystin/polyductin; FBD, FPC-binding domain; PC2BD, PC2-binding domain; HA, hemagglutinin; co-IP, co-immunoprecipitation. is characterized by numerous fluid-filled, spherical renal cysts, and autosomal recessive polycystic kidney disease (ARPKD) is characterized by massive, spindle-shaped renal cysts (1Igarashi P. Somlo S. J. Am. Soc. Nephrol. 2002; 13: 2384-2398Crossref PubMed Scopus (437) Google Scholar, 2Wilson P. N. Engl. J. Med. 2004; 350: 151-164Crossref PubMed Scopus (604) Google Scholar). 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El-Youssef M. Torres V.E. Harris P.C. Kidney Int. 2003; 64: 391-403Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 11Sharp A.M. Messiaen L.M. Page G. Antignac C. Gubler M.C. Onuchic L.F. Somlo S. Germino G.G. Guay-Woodford L.M. J. Med. Genet. 2005; 42: 336-349Crossref PubMed Scopus (76) Google Scholar). PKD1 has a 14-kb transcript and encodes PC1, a 4303-amino acid integral membrane protein with 11 putative transmembrane domains. PC1 is expressed in all tissues and organs of humans and mice (12American PKD1 ConsortiumHum. Mol. Genet. 1995; 4: 575-582Crossref PubMed Scopus (237) Google Scholar). The N-terminal region contains an extracellular portion of >3000 amino acids, which is predicted to be a site of protein-protein or receptor-ligand interactions (2Wilson P. N. Engl. J. Med. 2004; 350: 151-164Crossref PubMed Scopus (604) Google Scholar, 13Torres V.E. Harris P.C. Nat. Clin. Pract. Nephrol. 2006; 2: 40-55Crossref PubMed Scopus (235) Google Scholar). This region may also be released after cleavage at the GPS domain of PC1 and serve as a ligand for other proteins (14Yu S. Hackmann K. Gao J. He X. Piontek K. Garcia-Gonzalez M.A. Menezes L.F. Xu H. Germino G.G. Zuo J. Qian F. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 18688-18693Crossref PubMed Scopus (119) Google Scholar). The cytoplasmic C-terminal region of PC1 contains a putative coiled-coil domain that interacts with the PKD2 gene product, PC2 (15Qian F. Germino F.J. Cai Y. Zhang X. Somlo S. Germino G.G. Nat. Genet. 1997; 16: 179-183Crossref PubMed Scopus (549) Google Scholar). PKD2 has an ∼5.4-kb transcript and encodes PC2, a 968-amino acid protein that is predicted to be an integral membrane protein with six putative transmembrane domains and intracellular N and C termini (4Mochizuki T. Wu G. Hayashi T. Xenophontos S.L. Veldhuisen B. Saris J.J. Reynolds D.M. Cai Y. Gabow P.A. Pierides A. Kimberling W.J. Breuning M.H. Deltas C.C. Peters D.J. Somlo S. Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1149) Google Scholar). PC2 is a receptor-operated, nonselective cation channel; it is also known as TRPP2, a member of the trp superfamily (16Montell C. Birnbaumer L. Flockerzi V. Cell. 2002; 108: 595-598Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar, 17Qamar S. Vadivelu M. Sandford R. Biochem. Soc. Trans. 2007; 35: 124-128Crossref PubMed Scopus (28) Google Scholar). PKHD1 has a 16-kb transcript, contains at least 86 exons, and spans 470 kb on chromosome 6p12 (11Sharp A.M. Messiaen L.M. Page G. Antignac C. Gubler M.C. Onuchic L.F. Somlo S. Germino G.G. Guay-Woodford L.M. J. Med. Genet. 2005; 42: 336-349Crossref PubMed Scopus (76) Google Scholar). The longest open reading frame is predicted to be 66 exons and yields a 4074-amino acid type I membrane protein, FPC (5Ward C.J. Hogan M.C. Rossetti S. Walker D. Sneddon T. Wang X. Kubly V. Cunningham J.M. Bacallao R. Ishibashi M. Milliner D.S. Torres V.E. Harris P.C. Nat. Genet. 2002; 30: 259-269Crossref PubMed Scopus (574) Google Scholar, 6Onuchic L.F. Furu L. Nagasawa Y. Hou X. Eggermann T. Ren Z. Bergmann C. Senderek J. Esquivel E. Zeltner R. Rudnik-Schoneborn S. Mrug M. Sweeney W. Avner E.D. Zerres K. Guay-Woodford L.M. Somlo S. Germino G.G. Am. J. Hum. Genet. 2002; 70: 1305-1317Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 7Xiong H. Chen Y. Yi Y. Tsuchiya K. Moeckel G. Cheung J. Liang D. Tham K. Xu X. Chen X.-Z. Pei Y. Zhao Z.J. Wu G. Genomics. 2002; 80: 96-104Crossref PubMed Scopus (60) Google Scholar). The cytoplasmic C-terminal region of FPC is composed of 192 amino acids. FPC is predicted to be a membrane-associated, receptor-like protein. In addition, cleavage and release of its ectodomain into the renal tubular/duct lumen have been reported (5Ward C.J. Hogan M.C. Rossetti S. Walker D. Sneddon T. Wang X. Kubly V. Cunningham J.M. Bacallao R. Ishibashi M. Milliner D.S. Torres V.E. Harris P.C. Nat. Genet. 2002; 30: 259-269Crossref PubMed Scopus (574) Google Scholar, 18Kaimori J.Y. Nagasawa Y. Menezes L.F. Garcia-Gonzalez M.A. Deng J. Imai E. Onuchic L.F. Guay-Woodford L.M. Germino G.G. Hum. Mol. Genet. 2007; 16: 942-956Crossref PubMed Scopus (80) Google Scholar). Several recent reports have demonstrated functional and genetic relationships between FPC and the PCs (19Wu Y. Dai X.Q. Li Q. Chen C.X. Mai W. Hussain Z. Long W. Montalbetti N. Li G. Glynne R. Wang S. Cantiello H.F. Wu G. Chen X.-Z. Hum. Mol. Genet. 2006; 15: 3280-3292Crossref PubMed Scopus (98) Google Scholar, 20Garcia-Gonzalez M.A. Menezes L.F. Piontek K.B. Kaimori J. Huso D.L. Watnick T. Onuchic L.F. Guay-Woodford L.M. Germino G.G. Hum. Mol. Genet. 2007; 16: 1940-1950Crossref PubMed Scopus (86) Google Scholar, 21Wang S. Zhang J. Nauli S.M. Li X. Starremans P.G. Luo Y. Roberts K.A. Zhou J. Mol. Cell. Biol. 2007; 27: 3241-3252Crossref PubMed Scopus (141) Google Scholar, 22Kim I. Fu Y. Hui K. Moeckel G. Mai W. Li C. Liang D. Zhao P. Ma J. Chen X.-Z. George Jr., A.L. Coffey R.J. Feng Z.P. Wu G. J. Am. Soc. Nephrol. 2008; 19: 455-468Crossref PubMed Scopus (91) Google Scholar). Wu et al. (19Wu Y. Dai X.Q. Li Q. Chen C.X. Mai W. Hussain Z. Long W. Montalbetti N. Li G. Glynne R. Wang S. Cantiello H.F. Wu G. Chen X.-Z. Hum. Mol. Genet. 2006; 15: 3280-3292Crossref PubMed Scopus (98) Google Scholar) have demonstrated that KIF3B, a motor subunit of the heterotrimer kinesin-2, is able to bridge FPC and PC2 bindings. The activity of the PC2 channel is significantly altered when the binding complex is disrupted. It has also been reported that FPC regulates mechanotransduced Ca2+ responses, which may be induced by PC2 (21Wang S. Zhang J. Nauli S.M. Li X. Starremans P.G. Luo Y. Roberts K.A. Zhou J. Mol. Cell. Biol. 2007; 27: 3241-3252Crossref PubMed Scopus (141) Google Scholar), and that loss of FPC down-regulates PC2 expression in vivo (22Kim I. Fu Y. Hui K. Moeckel G. Mai W. Li C. Liang D. Zhao P. Ma J. Chen X.-Z. George Jr., A.L. Coffey R.J. Feng Z.P. Wu G. J. Am. Soc. Nephrol. 2008; 19: 455-468Crossref PubMed Scopus (91) Google Scholar). In addition, loss of FPC worsens the cystic phenotype of Pkd1 mutant mice, suggesting a genetic interaction between FPC and PC1 (20Garcia-Gonzalez M.A. Menezes L.F. Piontek K.B. Kaimori J. Huso D.L. Watnick T. Onuchic L.F. Guay-Woodford L.M. Germino G.G. Hum. Mol. Genet. 2007; 16: 1940-1950Crossref PubMed Scopus (86) Google Scholar). These findings imply a strong functional relationship between FPC and PCs in vivo, although the underlying molecular mechanisms are not fully understood. To explore the molecular relationships between these cystoproteins further, we studied the molecular interaction between PC2 and FPC. Through gene deletion and mutagenesis, we identified an FPC-binding domain (FBD) in the intracellular N terminus of PC2 and a PC2-binding domain (PC2BD) in the intracellular C terminus of FPC. This physical interaction prevents PC2 down-regulation induced by loss of FPC (22Kim I. Fu Y. Hui K. Moeckel G. Mai W. Li C. Liang D. Zhao P. Ma J. Chen X.-Z. George Jr., A.L. Coffey R.J. Feng Z.P. Wu G. J. Am. Soc. Nephrol. 2008; 19: 455-468Crossref PubMed Scopus (91) Google Scholar). Additionally, mice bearing a Neo-resistant cassette targeted to intron 2 of Pkd2, which leads to significant down-regulation of PC2, have a phenotype very similar to that of Pkhd1 mutant mice. This model suggests that the formation of spherical cysts, commonly observed in ADPKD, and the spindle-shaped dilatation of tubules, commonly observed in ARPKD, are induced by a common mechanism. These findings further define the roles for PC2 and FPC in the pathogenesis of both ADPKD and ARPKD and reveal a cystogenic link between ARPKD and ADPKD. Mouse Strains—We have previously reported the generation of the gene-targeted mouse model for Pkhd1 (Pkhd1–/–) (22Kim I. Fu Y. Hui K. Moeckel G. Mai W. Li C. Liang D. Zhao P. Ma J. Chen X.-Z. George Jr., A.L. Coffey R.J. Feng Z.P. Wu G. J. Am. Soc. Nephrol. 2008; 19: 455-468Crossref PubMed Scopus (91) Google Scholar). We have now generated a model in which a Cre-loxP/Flpe-FRT system was constructed as a Neo cassette flanked by two FRT sites (Neoflrt), and two loxP sites were inserted to flank exon 3 of Pkd2 (see Fig. 4, A and B). We found 206 embryonic stem cell colonies resistant to G418; one (W2A4) was selected after PCR screening using a pair of outside-construct and cassette-based primers. This cell line was confirmed by Southern blot analysis and injected into C57BL/6 blastocysts at the University of Connecticut Health Center Gene Targeting and Transgenic Facility. All mouse lines used in this study have been backcrossed onto the Bl6/C57 congenic background. Southern and Northern Blotting and Quantitative PCR—Southern analysis was used to genotype Pkhd1 and Pkd2 mutant mice using our published approaches (22Kim I. Fu Y. Hui K. Moeckel G. Mai W. Li C. Liang D. Zhao P. Ma J. Chen X.-Z. George Jr., A.L. Coffey R.J. Feng Z.P. Wu G. J. Am. Soc. Nephrol. 2008; 19: 455-468Crossref PubMed Scopus (91) Google Scholar, 42Wu G. Tian X. Cai Y. Markowitz G. D'Agati V. Park J.H. Yao L. Li L. Geng L. Zhao H. Edelmann W. Somlo S. Hum. Mol. Genet. 2002; 11: 1845-1854Crossref PubMed Scopus (108) Google Scholar). For Northern analysis, total RNA was isolated from embryos or kidneys using TRIzol reagent (Invitrogen) following the manufacturer's instructions. A probe (838 bp of exons 3–6 of Pkd2) was labeled using the RadPrime DNA labeling system (Invitrogen) with [α-32P]dCTP (PerkinElmer Life Sciences) and was hybridized with total RNA blots (25 μg/lane). Images of 28 S rRNA bands in these same blots were used as a total RNA loading control. Quantitative PCR was performed using the iCycler iQ real-time PCR detection system with the iQ SYBR Green Supermix kit (Bio-Rad). A pair of quantitative PCR primers was designed from the sequence of Pkd2 exon 6: 5′-GCG TGG TAC CCT CTT GGC AGT T-3′ (forward) and 5′-CAC GAC AAT CAC AAC ATC C-3′ (reverse). Antibodies—Polyclonalandmonoclonalantibodiesagainsthuman FPC (including hAR-C2p) and antibodies against human PC2 (hPKD2-Cp and hPKD2-Cm1A11, formerly designated PKD2A11) were described in our previous studies (19Wu Y. Dai X.Q. Li Q. Chen C.X. Mai W. Hussain Z. Long W. Montalbetti N. Li G. Glynne R. Wang S. Cantiello H.F. Wu G. Chen X.-Z. Hum. Mol. Genet. 2006; 15: 3280-3292Crossref PubMed Scopus (98) Google Scholar, 23Zhang M.Z. Mai W. Li C. Cho S.Y. Hao C. Moeckel G. Zhao R. Kim I. Wang J. Xiong H. Wang H. Sato Y. Wu Y. Nakanuma Y. Lilova M. Pei Y. Harris R.C. Li S. Coffey R.J. Sun L. Wu D. Chen X.-Z. Breyer M.D. Zhao Z.J. McKanna J.A. Wu G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2311-2316Crossref PubMed Scopus (136) Google Scholar). A polyclonal antibody against the intracellular N terminus of PC2 (Arg50– Val220), hPKD2-Np, was generated, and anti-PC2 specificity was confirmed (supplemental Fig. 1). The following antibodies and staining materials were purchased: anti-acetylatedα-tubulin, anti-γ-tubulin, anti-β-actin, anti-FLAG, and anti-hemagglutinin (HA) monoclonal antibodies (Sigma); fluorescein Lotus tetragonolobus lectin and fluorescein Dolichos biflorus agglutinin (Vector Laboratories); and fluorescein anti-Tamm-Horsfall glycoprotein (The Binding Site Ltd.). Western Blotting and Immunoprecipitation—Western analyses and immunoprecipitation experiments were performed using protocols similar to those described in our previous study (23Zhang M.Z. Mai W. Li C. Cho S.Y. Hao C. Moeckel G. Zhao R. Kim I. Wang J. Xiong H. Wang H. Sato Y. Wu Y. Nakanuma Y. Lilova M. Pei Y. Harris R.C. Li S. Coffey R.J. Sun L. Wu D. Chen X.-Z. Breyer M.D. Zhao Z.J. McKanna J.A. Wu G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2311-2316Crossref PubMed Scopus (136) Google Scholar). To avoid nonspecific binding in co-immunoprecipitation (co-IP), 0.05–3 m NaCl was used to wash the IP reaction. The entire intracellular termini and the deletion constructs of FPC and PC2 were constructed in FLAG-tagged and HA-tagged pCMV expression vectors (40Li Z. Hannigan M. Mo Z. Liu B. Lu W. Wu Y. Smrcka A.V. Wu G. Li L. Liu M. Huang C.K. Wu D. Cell. 2003; 114: 215-227Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar); their names and descriptions are listed in Fig. 1A and 2A. The FLAG-tagged entire intracellular C terminus of PC2 was designated PC2CT-F (amino acids 682–968).FIGURE 2Identification of the PC2BD of the intracellular C terminus of FPC.A, diagram of HA- and FLAG-tagged C-terminal fragments of FPC and an intracellular N-terminal fragment of PC2. Amino acid numbers defining each fragment are provided in parentheses. Co-IP results with PC2 are indicated with "+" for positive binding and "–" for no evidence of binding. B, co-IP of PC2NT-F with FCT-H, FCT1-H, or FCT2-H. Co-IP of PC2CT-F with FCT-H and single transfections of the above clones were used as negative controls. With anti-FLAG antibody for IP and anti-HA antibody for Western blotting, immunoreactivity indicates that PC2NT-F immunoprecipitated only with FCT-H (positive control) and FCT1-H. C, co-IP of PC2NT-F with FCT1-H, FCT4-H, FCT7-H, FCT6-H, FCT3-H, or FCT5-H. The left panel shows co-IP using anti-FLAG antibody for IP and anti-HA antibody for Western detection. Immunoreactivity indicates that FCT1-H, FCT4-H, FCT3-H, and FCT5-H immunoprecipitated with PC2NT-F, but FCT6-H and FCT7-H did not. Western blots show the level of cotransfected protein detected by anti-HA (upper right panel) and anti-FLAG (lower right panel) antibodies. D, co-IP using anti-PC2 polyclonal antibody (hPKD2-Np) for IP and anti-HA antibody for Western detection produces results similar to those in C. E, alignment of human (h) and mouse (m) FCT5-H, a candidate PC2BD; the mutagenized site in FCT-H (FCTMut-H) is shown. F, co-IP of PC2NT-F with FCT-H or FCTMut-H. The lower panel shows a Western blot of the levels of cotransfected protein detected by anti-HA antibody. The upper panel shows co-IP using hPKD2-Np for IP and anti-HA antibody for Western detection. Immunoreactivity indicates that FCT-H immunoprecipitated with PC2NT-F, but the mutagenized FCTMut-H did not.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Histology and Immunofluorescence Staining—Detailed procedures for histology and immunofluorescence were published previously (23Zhang M.Z. Mai W. Li C. Cho S.Y. Hao C. Moeckel G. Zhao R. Kim I. Wang J. Xiong H. Wang H. Sato Y. Wu Y. Nakanuma Y. Lilova M. Pei Y. Harris R.C. Li S. Coffey R.J. Sun L. Wu D. Chen X.-Z. Breyer M.D. Zhao Z.J. McKanna J.A. Wu G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2311-2316Crossref PubMed Scopus (136) Google Scholar). For microscopic analysis, images were obtained using a Zeiss Axioplan 2IE research microscope system with 4×, 10×, 20×, and 40× objectives. Cell Lines and Mouse Kidney Primary Epithelial Cell Cultures—All cell lines used in this study were cultured under previously described conditions (30Mai W. Chen D. Ding T. Kim I. Park S. Cho S. Chu J.S. Liang D. Wang N. Wu D. Li S. Zhao P. Zent R. Wu G. Mol. Biol. Cell. 2005; 16: 4398-4409Crossref PubMed Scopus (64) Google Scholar). The isolation and culture of primary renal epithelial cells from 2-month-old Pkhd1–/– and wild-type littermates were described in our previous study (22Kim I. Fu Y. Hui K. Moeckel G. Mai W. Li C. Liang D. Zhao P. Ma J. Chen X.-Z. George Jr., A.L. Coffey R.J. Feng Z.P. Wu G. J. Am. Soc. Nephrol. 2008; 19: 455-468Crossref PubMed Scopus (91) Google Scholar). Retrovirus Generation and Infection—The LZRS-ms-GFP system (a gift from Dr. Albert B. Reynolds, Vanderbilt University) was used to generate FPC fragment retroviruses (FCT-H and FCT1-H) and to transduce Pkhd1–/– cell pools. Cells were transduced by the retroviruses and collected after 1 day of culture for Western analysis (41Wildenberg G.A. Dohn M.R. Carnahan R.H. Davis M.A. Lobdell N.A. Settleman J. Reynolds A.B. Cell. 2006; 127: 1027-1039Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). Statistics—All biochemical assays were repeated two to three times. Statistical analysis was performed where appropriate using Student's t test or one-way analysis of variance followed by Tukey's multiple comparison test. Differences with p values <0.05 were considered statistically significant. Identification of an FBD in the Intracellular N Terminus of PC2—Recently, evidence for a physical interaction between the intracellular N terminus of PC2 and the C terminus of FPC was reported (22Kim I. Fu Y. Hui K. Moeckel G. Mai W. Li C. Liang D. Zhao P. Ma J. Chen X.-Z. George Jr., A.L. Coffey R.J. Feng Z.P. Wu G. J. Am. Soc. Nephrol. 2008; 19: 455-468Crossref PubMed Scopus (91) Google Scholar), but the precise nature of this interaction is unknown. Here, we applied a deletion-based strategy to identify the domains involved in the interaction between PC2 and FPC. First, we generated both FLAG- and HA-tagged constructs (FCT-F and FCT-H) that contain the entire intracellular portion of FPC. We also produced a series of HA-tagged deletion constructs of the intracellular N terminus of PC2 (Fig. 1A). We used two HA-tagged halves of the N terminus of PC2 (PC2NTa-H and PC2NTb-H) to perform co-IP experiments with the FLAG-tagged C terminus of FPC (FPC-F) cotransfected into HEK293 cells. Co-IP of the entire PC2 N terminus (PC2NT-H, HA-tagged) with FPC-F was used as a positive control (22Kim I. Fu Y. Hui K. Moeckel G. Mai W. Li C. Liang D. Zhao P. Ma J. Chen X.-Z. George Jr., A.L. Coffey R.J. Feng Z.P. Wu G. J. Am. Soc. Nephrol. 2008; 19: 455-468Crossref PubMed Scopus (91) Google Scholar). Positive immunoreactivity was observed for the positive control and the PC2NTa-H fragment, but not for the PC2NTb-H fragment (Fig. 1B), suggesting that the distal portion of the N terminus of PC2 (PC2NTb) is not involved in the FPC-PC2 interaction. To confirm, we singly transfected the same HA-tagged constructs and attempted to co-immunoprecipitate each construct with endogenous FPC using a polyclonal antibody specific for the intracellular C terminus of FPC (hAR-C2p) (23Zhang M.Z. Mai W. Li C. Cho S.Y. Hao C. Moeckel G. Zhao R. Kim I. Wang J. Xiong H. Wang H. Sato Y. Wu Y. Nakanuma Y. Lilova M. Pei Y. Harris R.C. Li S. Coffey R.J. Sun L. Wu D. Chen X.-Z. Breyer M.D. Zhao Z.J. McKanna J.A. Wu G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2311-2316Crossref PubMed Scopus (136) Google Scholar). These experiments confirmed that the proximal portion of the N terminus of PC2 (PC2NTa), but not the distal portion (PC2NTb), is involved in the binding between FPC and PC2 (Fig. 1C). To determine the minimum FBD of PC2, we generated a series of HA-tagged deletion constructs (PC2NTc–f-H) derived from PC2NTa (Fig. 1A) for co-IP with FPC-F. Positive immunoreactivity was observed for the positive control (PC2NT-H), PC2NTa-H, PC2NTc-H, and PC2NTe-H, but not for the PC2NTf-H fragment (Fig. 1, A and D). These data suggest that the amino acid fragment within the PC2NTe construct contains the FBD of PC2. Conversely, we used anti-HA antibody for co-IP and anti-FLAG antibody for Western detection and obtained the same results (data not shown). To confirm these results, we singly transfected the same HA-tagged constructs and attempted to co-immunoprecipitate each construct with endogenous FPC using hAR-C2p as described above (Fig. 1C). A similar result as in Fig. 1D was obtained (Fig. 1E). Together, these results indicate that Asp90–Arg139 of PC2 may function as an FBD. We used a mutagenic strategy to verify that the N terminus of PC2 binds FPC. We generated two mutagenic clones using the PC2NTc-H construct, placing two mutations within the FBD candidate region of PC2, respectively (Fig. 1F). The PC2NTcMut1-H construct harbors a mutation that replaces Ser122 with alanine (S122A), selected because Ser122 is a predicted protein kinase C phosphorylation site (4Mochizuki T. Wu G. Hayashi T. Xenophontos S.L. Veldhuisen B. Saris J.J. Reynolds D.M. Cai Y. Gabow P.A. Pierides A. Kimberling W.J. Breuning M.H. Deltas C.C. Peters D.J. Somlo S. Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1149) Google Scholar). The PC2NTcMut2-H construct harbors an R124A mutation, selected because the positively charged arginine may regulate the spatial conformation of the FBD. Co-IP experiments, as described previously, demonstrated that R124A significantly reduced FPC binding, but S122A did not (Fig. 1G). This provides additional evidence that the described FBD of PC2 binds to the intracellular C terminus of FPC. To finally confirm the location of the FBD within PC2, we generated a FLAG-tagged construct (PC2NTΔe-F) in which the candidate binding domain for FPC, Arg90–Arg139, was deleted in-frame (Fig. 1H). Co-IP experiments revealed that deletion of this region significantly diminished FPC binding to PC2 (Fig. 1I), suggesting that Arg90–Arg139 is the FBD of PC2. Identification of a PC2BD in the Intracellular Portion of FPC—Once the FBD of the intracellular N terminus of PC2 was determined, we used a similar strategy to identify the PC2BD of the intracellular C terminus of FPC. We used two HA-tagged halves of the C terminus of FPC (FCT1-H and FCT2-H) (Fig. 2A) to perform co-IP experiments with the FLAG-tagged N terminus of PC2 (PCNT-F) cotransfected into HEK293 cells. Co-IP of the entire FPC C terminus (FCT-H, HA-tagged) with PC2NT-F was used as a positive control. Cotransfection of the FLAG-tagged intracellular C terminus of PC2 (PC2CT-F) with FTC-H, as well as other single transfections, was used as a negative control (Fig. 2B). Positive immunoreactivity was observed for the positive control and the FCT1-H fragment, but not for the FCT2-H fragment or the negative controls (Fig. 2B). These data suggest that the distal half of the C terminus of FPC (FCT2-H) is not involved in the FPC-PC2 interaction. To determine the minimum PC2BD of FPC, we generated a series of HA-tagged deletion constructs (FCT3–7-H) within the intracellular C terminus of FPC (Fig. 2A) for co-IP with PC2NT-F. Positive immunoreactivity was observed for the positive control (FCT-H), FCT3-H, FCT4-H, and FCT5-H, but not for the FCT6-H or FCT7-H fragment (Fig. 2, A and C). These data suggest that FCT5-H is the minimum construct for the PC2BD of FPC. To confirm these results, we singly transfected the same HA-tagged constructs and attempted to co-immunoprecipitate each construct with endogenous PC2 using a polyclonal antibody specific for the intracellular C terminus of PC2 (hPKD2-Np) (Fig. 2D). Because the N terminus of PC2 bound to FCT5-H but not to FCT6-H or FCT7-H, Gln3903–Glu3963 may function as a PC2BD. Once again, we used a mutagenic strategy to verify that the candidate PC2BD binds PC2. We generated a construct (FCTMut-H) that harbors an R3931A missense mutation (Fig. 2E). The residue is positively charged and conserved in both human and mouse FPC. Neutralization of this residue was predicted to disrupt the structure of the PC2BD. FTC-H and FCTMut-H were each singly transfected; hPKD2-Np was used to co-immunoprecipitate endogenous PC2, and anti-HA antibody was used for detection. The R3931A mutation prevented FPC binding to PC2 (Fig. 2F), providing further evidence for physical interaction between the intracellular C terminus of FPC and the intracellular N terminus of PC2. FPC Binding to PC2 Prevents PC2 Down-regulation—We have previously reported that loss of FPC reduces PC2 expression in vivo (22Kim I. Fu Y. Hui K. Moeckel G. Mai W. Li C. Liang D. Zhao P. Ma J. Chen X.-Z. George Jr., A.L. Coffey R.J. Feng Z.P. Wu G. J. Am. Soc. Nephrol. 2008; 19: 455-468Crossref PubMed Scopus (91) Google Scholar), but the mechanism by which this occurs remains unknown. On the basis of the findings described above, we hypothesized that the physical interaction between the C terminus of FPC and the N terminus of PC2 is involved in the regulation of PC2 expression. First, we used primary cultures of renal epithelial cells derived from 2-month-old wild-type and Pkhd1–/– kidneys (22Kim I. Fu Y. Hui K. Moeckel G. Mai W. Li C. Liang D. Zhao P. Ma J. Chen X.-Z