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• W2059391323 abstract "p0071, a member of the armadillo protein family, localizes to both adherens junctions and desmosomes in epithelial cells and exhibits homology to the adherens junction protein p120 and the desmosomal protein plakophilin-1. p0071 is also present at dermal microvascular endothelial intercellular junctions and colocalizes with VE-cadherin, an endothelium-specific cadherin that associates with both actin and intermediate filament networks. To define the role of p0071 in junction assembly, p0071 was tested for interactions with other components of the endothelial junctional complex. In transient expression assays, p0071 colocalized with and formed complexes with both VE-cadherin and desmoplakin. Deletion analysis using the yeast two-hybrid system revealed that the armadillo repeat domain of p0071 bound directly to VE-cadherin. Site-directed mutagenesis experiments demonstrated that p0071 and p120 bound to the same region on the cytoplasmic tail of VE-cadherin and that overexpression of p0071 could displace p120 from intercellular junctions. In contrast to VE-cadherin, desmoplakin was found to associate with the non-armadillo head domain of p0071. Cotransfections and triple-label immunofluorescence analysis revealed that VE-cadherin colocalization with desmoplakin in transfected COS cells required p0071, suggesting that p0071 may couple VE-cadherin to desmoplakin. Based on previous findings that both VE-cadherin and desmoplakin play central roles in vasculogenesis, these new results suggest that p0071 may play an important role in endothelial junction assembly and in the morphogenic events associated with vascular remodeling. p0071, a member of the armadillo protein family, localizes to both adherens junctions and desmosomes in epithelial cells and exhibits homology to the adherens junction protein p120 and the desmosomal protein plakophilin-1. p0071 is also present at dermal microvascular endothelial intercellular junctions and colocalizes with VE-cadherin, an endothelium-specific cadherin that associates with both actin and intermediate filament networks. To define the role of p0071 in junction assembly, p0071 was tested for interactions with other components of the endothelial junctional complex. In transient expression assays, p0071 colocalized with and formed complexes with both VE-cadherin and desmoplakin. Deletion analysis using the yeast two-hybrid system revealed that the armadillo repeat domain of p0071 bound directly to VE-cadherin. Site-directed mutagenesis experiments demonstrated that p0071 and p120 bound to the same region on the cytoplasmic tail of VE-cadherin and that overexpression of p0071 could displace p120 from intercellular junctions. In contrast to VE-cadherin, desmoplakin was found to associate with the non-armadillo head domain of p0071. Cotransfections and triple-label immunofluorescence analysis revealed that VE-cadherin colocalization with desmoplakin in transfected COS cells required p0071, suggesting that p0071 may couple VE-cadherin to desmoplakin. Based on previous findings that both VE-cadherin and desmoplakin play central roles in vasculogenesis, these new results suggest that p0071 may play an important role in endothelial junction assembly and in the morphogenic events associated with vascular remodeling. Vascular endothelial cells form a continuous cell layer along the wall of blood vessels and participate in a wide range of biological processes that regulate vascular function. One important function of the endothelial lining is to control the movement of solutes and fluid from the vascular space to the tissues (1Lum H. Malik A.B. Can. J. Physiol. Pharmacol. 1996; 74: 787-800PubMed Google Scholar, 2Stevens T. Garcia J.G. Shasby D.M. Bhattacharya J. Malik A.B. Am. J. Physiol. 2000; 279: L419-L422Crossref PubMed Google Scholar). The loss of cell adhesion between endothelial cells results in tissue edema, inflammation, and poor wound healing. In addition, these cell-cell contacts also function as plasma membrane attachment sites for cytoskeletal networks, such as actin and intermediate filaments, thereby influencing cell shape and tissue integrity (3Kowalczyk A.P. Bornslaeger E.A. Norvell S.M. Palka H.L. Green K.J. Int. Rev. Cytol. 1999; 185: 237-302Crossref PubMed Google Scholar, 4Dejana E. Bazzoni G. Lampugnani M.G. Exp. Cell Res. 1999; 252: 13-19Crossref PubMed Scopus (211) Google Scholar, 5Lampugnani M.G. Dejana E. Curr. Opin. Cell Biol. 1997; 9: 674-682Crossref PubMed Scopus (199) Google Scholar). It is now apparent that intercellular junctions are macromolecular complexes that integrate informational cues derived from cell adhesion events with intracellular signaling pathways that regulate cell proliferation, apoptosis, and gene expression (6Klymkowsky M.W. Parr B. Cell. 1995; 83: 5-8Abstract Full Text PDF PubMed Scopus (109) Google Scholar, 7Barth A.I. Nathke I.S. Nelson W.J. Curr. Opin. Cell Biol. 1997; 9: 683-690Crossref PubMed Scopus (488) Google Scholar). The mechanisms by which adhesive interactions are established between vascular endothelial cells have been studied extensively (8Dejana E. J. Clin. Invest. 1996; 98: 1949-1953Crossref PubMed Scopus (272) Google Scholar). Endothelial cells express a unique cadherin, VE-cadherin (where VE is vascular endothelial; cadherin-5), which plays a central role in the establishment and maintenance of endothelial monolayer integrity and angiogenesis (9Breviario F. Caveda L. Corada M. Martin-Padura I. Navarro P. Golay J. Introna M. Gulino D. Lampugnani M.G. Dejana E. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1229-1239Crossref PubMed Scopus (241) Google Scholar, 10Liao F., Li, Y. O'Connor W. Zanetta L. Bassi R. Santiago A. Overholser J. Hooper A. Mignatti P. Dejana E. Hicklin D.J. Bohlen P. Cancer Res. 2000; 60: 6805-6810PubMed Google Scholar). Significantly, endothelial cells assemble unique junctional complexes that couple VE-cadherin to both actin and intermediate filament networks (11Kowalczyk A.P. Navarro P. Dejana E. Bornslaeger E.A. Green K.J. Kopp D.S. Borgwardt J.E. J. Cell Sci. 1998; 111: 3045-3057Crossref PubMed Google Scholar), although the precise nature of these protein interactions remains only partially characterized. Like other classical cadherins such as E-cadherin, VE-cadherin associates with the cytoplasmic proteins β-catenin and plakoglobin, members of the armadillo protein family (12Lampugnani M.G. Corada M. Caveda L. Breviario F. Ayalon O. Geier B. Dejana E. J. Cell Biol. 1995; 129: 203-217Crossref PubMed Scopus (497) Google Scholar). Both plakoglobin and β-catenin bind directly to the cytoplasmic domain of VE-cadherin, thus providing a link to the actin cytoskeleton through their association with α-catenin, a vinculin homolog that plays a key role in linking the cadherin complex to the actin cytoskeleton. In addition, plakoglobin (but not β-catenin) provides a link from VE-cadherin to the intermediate filament network by recruiting desmoplakin, an intermediate filament-binding protein, to intercellular junctions (11Kowalczyk A.P. Navarro P. Dejana E. Bornslaeger E.A. Green K.J. Kopp D.S. Borgwardt J.E. J. Cell Sci. 1998; 111: 3045-3057Crossref PubMed Google Scholar). Desmoplakin, a member of the plakin family of cytoskeletal cross-linking proteins, is an abundant constituent of the desmosomal plaque and functions to couple intermediate filaments to membrane-associated adhesive junctions (13Leung C.L. Liem R.K. Parry D.A. Green K.J. J. Cell Sci. 2001; 114: 3409-3410PubMed Google Scholar, 14Leung C.L. Green K.J. Liem R.K. Trends Cell Biol. 2002; 12: 37-45Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). Previous studies demonstrated that the carboxyl terminus of desmoplakin interacts directly with intermediate filament networks (15Stappenbeck T.S. Green K.J. J. Cell Biol. 1992; 116: 1197-1209Crossref PubMed Scopus (159) Google Scholar, 16Stappenbeck T.S. Bornslaeger E.A. Corcoran C.M. Luu H.H. Virata M.L.A. Green K.J. J. Cell Biol. 1993; 123: 691-705Crossref PubMed Scopus (150) Google Scholar, 17Kouklis P.D. Hutton E. Fuchs E. J. Cell Biol. 1994; 127: 1049-1060Crossref PubMed Scopus (243) Google Scholar, 18Meng J.-J. Bornslaeger E.A. Green K.J. Steinert P.M. Ip W. J. Biol. Chem. 1997; 272: 21495-21503Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), whereas the amino terminus of desmoplakin associates with desmosomes through interactions with plakoglobin (19Kowalczyk A.P. Bornslaeger E.A. Borgwardt J.E. Palka H.L. Dhaliwal A.S. Corcoran C.M. Denning M.F. Green K.J. J. Cell Biol. 1997; 139: 773-784Crossref PubMed Scopus (196) Google Scholar, 20Smith E.A. Fuchs E. J. Cell Biol. 1998; 141: 1229-1241Crossref PubMed Scopus (205) Google Scholar) and the plakophilins (21Kowalczyk A.P. Hatzfeld M. Bornslaeger E.A. Kopp D.S. Borgwardt J.E. Corcoran C.M. Settler A. Green K.J. J. Biol. Chem. 1999; 274: 18145-18148Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 22Hatzfeld M. Haffner C. Schulze K. Vinzens U. J. Cell Biol. 2000; 149: 209-222Crossref PubMed Scopus (138) Google Scholar, 23Chen X. Bonne S. Hatzfeld M. Van Roy F. Green K.J. J. Biol. Chem. 2002; 277: 10512-10522Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). In addition to playing fundamental roles in desmosome assembly and epidermal integrity (24Vasioukhin V. Bowers E. Bauer C. Degenstein L. Fuchs E. Nat. Cell. Biol. 2001; 3: 1076-1085Crossref PubMed Scopus (248) Google Scholar), desmoplakin also plays important roles in adherens junction formation (24Vasioukhin V. Bowers E. Bauer C. Degenstein L. Fuchs E. Nat. Cell. Biol. 2001; 3: 1076-1085Crossref PubMed Scopus (248) Google Scholar, 25Hatsell S. Cowin P. Nat. Cell. Biol. 2001; 3: E270-E272Crossref PubMed Scopus (39) Google Scholar). Interestingly, vascular and lymphatic endothelial cells, which do not assemble true desmosomes, also express desmoplakin (26Schmelz M. Franke W.W. Eur. J. Cell Biol. 1993; 61: 274-289PubMed Google Scholar, 27Schmelz M. Moll R. Kuhn C. Franke W.W. Differentiation. 1994; 57: 97-117Crossref PubMed Scopus (101) Google Scholar). In cultured human umbilical vascular endothelial cells and primary dermal microvascular endothelial cells, desmoplakin was shown to localize to intercellular junctions (11Kowalczyk A.P. Navarro P. Dejana E. Bornslaeger E.A. Green K.J. Kopp D.S. Borgwardt J.E. J. Cell Sci. 1998; 111: 3045-3057Crossref PubMed Google Scholar, 28Valiron O. Chevier V. Usson Y. Breviario F. Job D. Dejana E. J. Cell Sci. 1996; 109: 2141-2149Crossref PubMed Google Scholar). Recent studies also indicate that desmoplakin plays an important role in vasculogenesis (29Gallicano G.I. Bauer C. Fuchs E. Development. 2001; 128: 929-941PubMed Google Scholar), underscoring the need to determine how desmoplakin interacts with other components of endothelial intercellular junctions. An exciting development over the last several years is the realization that intercellular junctions contain a newly identified subset of armadillo family proteins termed the p120/plakophilin subfamily (30Hatzfeld M. Int. Rev. Cytol. 1999; 186: 179-224Crossref PubMed Google Scholar,31Anastasiadis P.Z. Reynolds A.B. J. Cell Sci. 2000; 113: 1319-1334Crossref PubMed Google Scholar). In general, p120 assembles into actin-associated adherens junctions, whereas the plakophilins assemble into intermediate filament-based desmosomes. Interestingly, one member of the subfamily (termed p0071) assembles into both adherens junctions and desmosomes (32Hatzfeld M. Nachtsheim C. J. Cell Sci. 1996; 109: 2767-2778Crossref PubMed Scopus (127) Google Scholar). This dual targeting of p0071 to both types of junctions raises the possibility that p0071 plays a role in the molecular cross-talk that occurs between different intercellular junctions, thus influencing a range of cellular events that are regulated by either the actin or intermediate filament cytoskeleton. To understand how p0071 might perform specific roles in junction assembly, we initiated an investigation into the subcellular localization of p0071 in dermal microvascular endothelial cells. In addition, we tested p0071 for interactions with other intercellular junction proteins using transient transfection of COS-7 and 293 cells, co-immunoprecipitation, and yeast two-hybrid analysis. The data indicate that p0071 associates with both adherens junction and desmosomal proteins and that p0071 is a cadherin- and desmoplakin-binding protein. A cDNA construct encoding full-length human p0071 was generated as described previously (32Hatzfeld M. Nachtsheim C. J. Cell Sci. 1996; 109: 2767-2778Crossref PubMed Scopus (127) Google Scholar) and subcloned into theNotI site of the pKS vector (Stratagene, La Jolla, CA). Expression in eukaryotic cells was performed with the FLAG-tagged expression system pCMV-Tag4 or pCMV-Tag2 vector, containing a carboxyl- or an amino-terminal FLAG epitope, respectively (Stratagene). p0071-(1–1016)-FLAG was constructed using the 3.0-kbNotI/SalI fragment from the pKS vector containing the p0071 insert and subcloned into pCMV-Tag4. Similarly, p0071-(1–553)-FLAG was generated from a 1.8-kbNotI/PstI fragment and subcloned into pCMV-Tag4. p0071-(554–1016)-FLAG was generated from a 1.8-kbPstI/SalI fragment and subcloned into the pCMV-Tag2 vector. To generate full-length FLAG-tagged p0071 (p0071-(1–1192)-FLAG), a point mutation was introduced to eliminate the stop codon using the primer pair 5′-GAC TCA TGG GTG GCG GAT CAA GCT TCC CAA CAG AGG and 5′-CCT CTG TTG GGA AGC TTG ATC CGC CAC CCA TGA GTC and the QuikChangeTM site-directed mutagenesis kit (Stratagene). The resulting 3.5-kb BamHI/HindIII fragment was subcloned into the pCMV-Tag4 vector. The p0071-(1–508)-FLAG construct was generated by PCR using a p0071 5′-primer (5′-CGC GGATCC AGA GGA ATG CCA GCT CCT GAG CAG GCC), which generates a BamHI site (underlined) at the 5′-end of p0071-(1–508)-FLAG, and the p0071-(1–508)-FLAG 3′-primer (5′-TTT AAC GTCGAC CTA CTT ATC GTC GTC ATC CTT GTA ATC GGT GCC ATC ATC AGC CGG CAC), which generates the FLAG tag (italicized) and a 3′-SalI site (underlined). p0071-(509–992)-FLAG was also generated using a p0071 5′-primer (5′-CGC GGATCC AGA GGA ATG ACA AGA TCC CCA TCA ATA GAC), which generates a BamHI site (underlined) at the 5′-end of the construct, and the p0071-(509–992)-FLAG 3′-primer (5′-CCG CTCGAGCTA CTT ATC GTC GTC ATC CTT GTA ATC CCA TAA TGT ATT CAA GAC CTG GGC), which generates the FLAG tag (italicized) and a 3′-XhoI site (underlined). p0071-(992–1192)-FLAG was generated by PCR using a p0071 5′-primer (5′-CGC GGATCC AGA GGA ATG CAA TAT CGG GAC CTC CGG AGC), which generates a BamHI site (underlined) at the 5′-end of p0071-(992–1192)-FLAG, and the p0071-(992–1192)-FLAG 3′ primer (5′-CCG CTCGAG CTA CTT ATC GTC GTC ATC CTT GTA ATC CAC CCA TGA GTC TGG GGA CCC), which generates a FLAG tag (italicized) and a 3′-XhoI site (underlined). The PCR products were subcloned into donor vectors of the Creator system (Clontech, Palo Alto, CA), and PCR-generated regions were sequenced to verify that errors were not introduced into the coding sequence. Expression of all constructs was further verified by Western blotting and immunofluorescence. A diagram illustrating the different domains of the recombinant proteins used in this work is shown in Fig. 1. Full-length cDNA encoding human VE-cadherin, subcloned into the pECE vector using the SV40 promoter, was generated as described previously (9Breviario F. Caveda L. Corada M. Martin-Padura I. Navarro P. Golay J. Introna M. Gulino D. Lampugnani M.G. Dejana E. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1229-1239Crossref PubMed Scopus (241) Google Scholar, 33Navarro P. Caveda L. Breviario F. Mandoteanu I. Lampugnani M.G. Dejana E. J. Biol. Chem. 1995; 270: 30965-30972Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). The cDNA clone encoding the cytoplasmic domain of human VE-cadherin was generated by PCR as described previously (34Venkiteswaran K. Xiao K. Summers S. Calkins C.C. Vincent P.A. Pumiglia K. Kowalczyk A.P. Am. J. Physiol. 2002; 283: C811-C821Crossref PubMed Scopus (105) Google Scholar). The cytoplasmic domain was recovered from the TA vector (Invitrogen) by restriction digestion withEcoRI/XhoI and subcloned into the Creator system donor vector. To generate the triple-alanine mutation in the VE-cadherin juxtamembrane domain (E652A/M653A/D654A), we used the QuikChange site-directed mutagenesis kit and the primer pair 5′-GAG GGC GGC GGC GCG GCG GCC ACC ACC AGC TAC G and 5′-CGT AGC TGG TGG TGG CCG CCG CGC CGC CGC CCT C. cDNAs encoding full-length desmoplakin with a carboxyl-terminal Myc epitope tag, the first 584 amino acids of desmoplakin (DPNTP, for desmoplakinN-terminal polypeptide) (35Bornslaeger E.A. Corcoran C.M. Stappenbeck T.S. Green K.J. J. Cell Biol. 1996; 134: 985-1001Crossref PubMed Scopus (182) Google Scholar), and a truncated desmoplakin polypeptide lacking the amino-terminal domain (DPΔN) (15Stappenbeck T.S. Green K.J. J. Cell Biol. 1992; 116: 1197-1209Crossref PubMed Scopus (159) Google Scholar, 16Stappenbeck T.S. Bornslaeger E.A. Corcoran C.M. Luu H.H. Virata M.L.A. Green K.J. J. Cell Biol. 1993; 123: 691-705Crossref PubMed Scopus (150) Google Scholar) were generously provided by Dr. K. J. Green (Fig. 1). Yeast two-hybrid vectors encoding the Gal4 DNA-binding domain (pLP-GBK) or transcription activation domain (pLP-GAD) were purchased from Clontech. The p0071 constructs described above were subcloned from the Creator system donor vector into the Creator system acceptor vector pLP-GBK. The VE-cadherin cytoplasmic domain was subcloned into the Creator system donor vector, followed by recombination subcloning into the acceptor vector pLP-GAD. All constructs were then verified by sequencing. To assay for interactions between proteins, 5–10 μg of plasmid DNA was transformed into the yeast strain AH109 (Clontech) using LiAc, and double transformants were selected by growth in the absence of leucine and tryptophan. Expression of the HIS andADE reporter genes was analyzed by monitoring colony growth on plates lacking histidine, adenine, leucine, and tryptophan. The monkey kidney cell line COS-7 and the human embryonic kidney cell line 293 were routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT) and penicillin/streptomycin/amphotericin B (Invitrogen). Primary cultures of human dermal microvascular endothelial cells were purchased from the Emory Skin Diseases Research Center (Core B) and cultured in MDCB131 (Invitrogen) supplemented with 10% fetal bovine serum, 100 μg/ml cAMP (Sigma), 1 μg/ml hydrocortisone (Sigma), 10 ng/ml epidermal growth factor (Intergen Co., Purchase, NY), and penicillin/streptomycin/amphotericin B (36Muller W.A. Giambrone M.A., Jr. J. Cell Biol. 1986; 103: 2389-2402Crossref PubMed Scopus (65) Google Scholar). For transient transfection experiments, a subclone of COS-7 cells (COS-7-20) was transfected by calcium phosphate precipitation. The cells were fixed and processed for immunofluorescence analysis after 24–48 h. The distribution of p0071 in microvascular endothelial cells and transiently transfected COS-7 cells was analyzed by immunofluorescence. Cells grown on coverslips were rinsed in phosphate-buffered saline and fixed in methanol at −20 °C for 4 min. Alternatively, cells were fixed in 3.7% paraformaldehyde in phosphate-buffered saline, followed by permeabilization in 0.5% Triton X-100 in phosphate-buffered saline. VE-cadherin was monitored using mouse anti-cadherin-5 monoclonal antibody (Transduction Laboratories, Lexington, KY). Desmoplakin was detected using rabbit polyclonal antibody NW6, directed against the desmoplakin carboxyl-terminal domain (37Angst B.D. Nilles L.A. Green K.J. J. Cell Sci. 1990; 97: 247-257Crossref PubMed Google Scholar), or the Myc epitope tag (Bethyl Laboratories, Inc., Montgomery, TX). Endogenous p0071 was analyzed with a rabbit polyclonal antibody (32Hatzfeld M. Nachtsheim C. J. Cell Sci. 1996; 109: 2767-2778Crossref PubMed Scopus (127) Google Scholar), whereas FLAG-tagged p0071 was detected by an anti-FLAG tag monoclonal antibody (Stratagene). Appropriate species cross-absorbed secondary antibodies conjugated to rhodamine, fluorescein, or various Alexa Fluors (Molecular Probes, Inc., Eugene, OR) were used for dual-label immunofluorescence. Control experiments were carried out routinely to verify that fluorescence was not due to secondary antibody cross-reactivity. A Leica DMR-E fluorescence microscope equipped with narrow band-pass filters and a Hamamatsu Orca camera was used. Images were captured and processed using Open Lab software (Improvision, Inc., Lexington, MA). Immunoprecipitation was carried out as described previously (19Kowalczyk A.P. Bornslaeger E.A. Borgwardt J.E. Palka H.L. Dhaliwal A.S. Corcoran C.M. Denning M.F. Green K.J. J. Cell Biol. 1997; 139: 773-784Crossref PubMed Scopus (196) Google Scholar,21Kowalczyk A.P. Hatzfeld M. Bornslaeger E.A. Kopp D.S. Borgwardt J.E. Corcoran C.M. Settler A. Green K.J. J. Biol. Chem. 1999; 274: 18145-18148Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Briefly, cells were scraped into Tris-buffered saline containing 0.5% Triton X-100, vortexed, and subjected to centrifugation at 14,000 × g. Antibody M2 (directed against the FLAG tag) conjugated to agarose beads (Sigma) was incubated with the cell lysate for 1 h at 4 °C. Immune complexes were captured by centrifugation, and the beads were washed four times in Tris-buffered saline containing 0.5% Triton X-100 for 10 min with gentle rotation at 4 °C. Immune complexes were released by incubation in reducing SDS-PAGE sample buffer at 95 °C and then separated on 7.5% acrylamide gels and transferred to nitrocellulose according to standard protocols. Membranes were washed and incubated with horseradish peroxidase-coupled secondary antibodies, and bound antibodies were visualized by chemiluminescence reagent (AmershamBiosciences). The precise localization of p0071 in different cell types and the mechanisms by which p0071 incorporates into junctions have been only partially characterized. p0071 has been shown previously to localize at cell-cell contacts in epithelial compartments of various tissues (32Hatzfeld M. Nachtsheim C. J. Cell Sci. 1996; 109: 2767-2778Crossref PubMed Scopus (127) Google Scholar, 38Schroder R. van der Ven P.F. Warlo I. Schumann H. Furst D.O. Blumcke I. Schmidt M.C. Hatzfeld M. J. Muscle Res. Cell Motil. 2000; 21: 577-586Crossref PubMed Scopus (6) Google Scholar) and was found to be expressed and assembled into intercellular junctions of a variety of cultured epithelial cell lines, including HeLa, A431, and HaCaT (32Hatzfeld M. Nachtsheim C. J. Cell Sci. 1996; 109: 2767-2778Crossref PubMed Scopus (127) Google Scholar). Using previously characterized antibodies specific for p0071 (32Hatzfeld M. Nachtsheim C. J. Cell Sci. 1996; 109: 2767-2778Crossref PubMed Scopus (127) Google Scholar), the distribution of p0071 was examined and compared with that of VE-cadherin in cultured primary human microvascular endothelial cells. In confluent monolayers of primary microvascular endothelial cells, p0071 localized at intercellular junctions (Fig. 2 A) and colocalized with VE-cadherin (Fig. 2, C and D). These results are consistent with previous studies indicating that p0071 is broadly expressed and assembled into intercellular junctions in a variety of cell types and tissues (32Hatzfeld M. Nachtsheim C. J. Cell Sci. 1996; 109: 2767-2778Crossref PubMed Scopus (127) Google Scholar). The localization of p0071 at cell-cell borders suggested that this protein may interact with other components within the intercellular junctions of microvascular endothelial cells, such as VE-cadherin. To begin the analysis of p0071 binding partners, a FLAG-tagged p0071 construct (p0071-(1–1016)-FLAG) was generated. When expressed in COS-7 cells, the p0071-(1–1016)-FLAG protein localized to both the cytoplasm and intercellular junctions (Fig. 3 B). To determine whether p0071 interacts with VE-cadherin, p0071-(1–1016)-FLAG was cotransfected with VE-cadherin (Fig. 3, C and D). p0071-(1–1016)-FLAG (Fig. 3 C) and VE-cadherin (Fig. 3 D) exhibited extensive colocalization, primarily at cell-cell borders. Moreover, in cells coexpressing p0071-(1–1016)-FLAG and VE-cadherin, p0071 recruitment to intercellular junctions was dramatically increased. The central armadillo domain of p0071, but not the non-armadillo head domain, colocalized with VE-cadherin when cotransfected into COS-7 cells (data not shown), suggesting that the armadillo domain of p0071 binds to VE-cadherin. To test for direct interactions between p0071 and VE-cadherin, full-length p0071 and the cytoplasmic domain of VE-cadherin were examined for direct binding using the yeast two-hybrid system. Direct interactions between proteins were assayed by growth in the absence of histidine and adenine (Fig. 3 E). In agreement with the COS-7 cell transient transfection assays, growth in the absence of histidine and adenine was consistently observed when yeast were cotransformed with plasmids encoding p0071 in the DNA-binding domain vector and the VE-cadherin cytoplasmic domain in the transcription activation domain vector (Fig. 3 E). Similar results were obtained using β-galactosidase as a reporter for direct interactions (data not shown), indicating that p0071 and VE-cadherin are direct binding partners. To further define the domain of p0071 that binds to VE-cadherin, deletion mutants of p0071 (p0071-(1–508), p0071-(509–992), and p0071-(324–1192)) were generated and analyzed for the ability to bind to the VE-cadherin cytoplasmic domain (Fig. 3 E). The results indicate that the central armadillo domain of p0071 (amino acids 509–992) is necessary and sufficient for binding to the cytoplasmic domain of VE-cadherin. These observations are consistent with the hypothesis that, like the related proteins ARVCF (a rmadillo repeat gene deleted in velo cardiofacial syndrome), δ-catenin, and p120, p0071 is a cadherin-binding protein. Recent studies have identified a core region within the juxtamembrane domain of E-cadherin responsible for binding to the armadillo protein p120 (39Thoreson M.A. Anastasiadis P.Z. Daniel J.M. Ireton R.C. Wheelock M.J. Johnson K.R. Hummingbird D.K. Reynolds A.B. J. Cell Biol. 2000; 148: 189-202Crossref PubMed Scopus (389) Google Scholar). The juxtamembrane domain sequence is highly conserved among type I and II classical cadherins (31Anastasiadis P.Z. Reynolds A.B. J. Cell Sci. 2000; 113: 1319-1334Crossref PubMed Google Scholar). To test whether p0071 and p120 compete for binding sites at cell-cell junctions, full-length FLAG-tagged p0071 was transiently expressed in COS-7 cells. The cells were then processed for immunofluorescence microscopy using antibodies directed against the FLAG epitope tag to detect exogenously expressed p0071 along with antibodies directed against endogenous p120. In untransfected cells, p120 was detected in a continuous pattern along cell-cell contacts (Fig. 4, Aand B). In contrast, p120 staining was dramatically reduced at the borders of adjacent cells expressing the FLAG-tagged p0071 protein (Fig. 4, C and D), suggesting that p0071 occupies membrane-binding sites in a mutually exclusive manner with p120. To determine whether p0071 and p120 bind to the same region of VE-cadherin, a triple-alanine substitution at residues 652–654 of the VE-cadherin juxtamembrane domain was generated and tested for the ability to bind to plakoglobin, p0071, or p120 in the yeast two-hybrid system. Protein-protein interactions were monitored by growth in the absence of histidine and adenine. Although plakoglobin bound to the mutated VE-cadherin cytoplasmic domain, neither p0071 (Fig. 4 E) nor p120 (data not shown) interacted with the mutated cytoplasmic tail of VE-cadherin. Similar results were obtained when full-length VE-cadherin with the E652A/M653A/D654A mutation was expressed in COS-7 cells; the VE-cadherin mutant failed to colocalize with p0071 as observed by dual-label immunofluorescence (data not shown). These data indicate that p0071 binds to the juxtamembrane domain of VE-cadherin and that it may compete with p120 for binding to this highly conserved region of cadherins. Vascular endothelial cells assemble unique intercellular junctions that are thought to couple VE-cadherin not only to actin microfilaments, but also to the vimentin intermediate filament cytoskeleton. Several studies have demonstrated that the intermediate filament-binding protein desmoplakin is expressed in endothelial cells and assembled into endothelial intercellular junctions (26Schmelz M. Franke W.W. Eur. J. Cell Biol. 1993; 61: 274-289PubMed Google Scholar, 27Schmelz M. Moll R. Kuhn C. Franke W.W. Differentiation. 1994; 57: 97-117Crossref PubMed Scopus (101) Google Scholar). Furthermore, desmoplakin and VE-cadherin have been shown to co-assemble into intercellular junctions of cultured endothelial cells (11Kowalczyk A.P. Navarro P. Dejana E. Bornslaeger E.A. Green K.J. Kopp D.S. Borgwardt J.E. J. Cell Sci. 1998; 111: 3045-3057Crossref PubMed Google Scholar, 28Valiron O. Chevier V. Usson Y. Breviario F. Job D. Dejana E. J. Cell Sci. 1996; 109: 2141-2149Crossref PubMed Google Scholar), and recent studies indicate that desmoplakin plays a central role in blood vessel formation during development (29Gallicano G.I. Bauer C. Fuchs E. Development. 2001; 128: 929-941PubMed Google Scholar). Based on these previous reports and on the observation that p0071 localizes to both adherens junctions and desmosomes (32Hatzfeld M. Nachtsheim C. J. Cell Sci. 1996; 109: 2767-2778Crossref PubMed Scopus (127) Google Scholar), p0071 was tested for interactions with desmoplakin. p0071-(1–1016)-FLAG and full-length Myc-tagged desmoplakin were coexpressed in COS-7 cells (Fig. 5). In tran" @default.
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