Title: Characterization of Neuropilin-1 Structural Features That Confer Binding to Semaphorin 3A and Vascular Endothelial Growth Factor 165
Abstract: Neuropilin-1 (Npn-1) is a receptor for both semaphorin 3A (Sema3A) and vascular endothelial growth factor 165 (VEGF165). To understand the role Npn-1 plays as a receptor for these structurally and functionally unrelated ligands, we set out to identify structural features of Npn-1 that confer binding to Sema3A or VEGF165. We constructed Npn-1 variants containing deletions within the “a” and “b” domains of Npn-1. More than 16 variants were expressed in COS-1 cells and tested for alkaline phosphatase-Sema3A as well as alkaline phosphatase-VEGF165binding. Our results indicate that each of the two Npn-1 CUB domains and the amino-terminal coagulation factor V/VIII domain (CF V/VIII) are essential for Sema3A binding, but only the amino-terminal Npn-1 CF V/VIII domain is required for binding to VEGF165. Guided by the structure of the bovine spermadhesin CUB domain, point mutants targeting defined surfaces of the Npn-1 a1 CUB domain were generated and tested for Sema3A and VEGF165 binding. One Npn-1 variant, Npn-12ABC, exhibits complete loss of Sema3A binding while retaining normal VEGF165 binding. Moreover, co-immunoprecipitation experiments show that Npn-12ABC can form a signaling complex with the VEGF165 signaling receptor KDR/VEGFR-2. These results establish the identity of contact sites between Npn-1 and its semaphorin ligands, and they provide a foundation for understanding how Npn-1 functions as a receptor for distinct classes of ligands in vivo. Neuropilin-1 (Npn-1) is a receptor for both semaphorin 3A (Sema3A) and vascular endothelial growth factor 165 (VEGF165). To understand the role Npn-1 plays as a receptor for these structurally and functionally unrelated ligands, we set out to identify structural features of Npn-1 that confer binding to Sema3A or VEGF165. We constructed Npn-1 variants containing deletions within the “a” and “b” domains of Npn-1. More than 16 variants were expressed in COS-1 cells and tested for alkaline phosphatase-Sema3A as well as alkaline phosphatase-VEGF165binding. Our results indicate that each of the two Npn-1 CUB domains and the amino-terminal coagulation factor V/VIII domain (CF V/VIII) are essential for Sema3A binding, but only the amino-terminal Npn-1 CF V/VIII domain is required for binding to VEGF165. Guided by the structure of the bovine spermadhesin CUB domain, point mutants targeting defined surfaces of the Npn-1 a1 CUB domain were generated and tested for Sema3A and VEGF165 binding. One Npn-1 variant, Npn-12ABC, exhibits complete loss of Sema3A binding while retaining normal VEGF165 binding. Moreover, co-immunoprecipitation experiments show that Npn-12ABC can form a signaling complex with the VEGF165 signaling receptor KDR/VEGFR-2. These results establish the identity of contact sites between Npn-1 and its semaphorin ligands, and they provide a foundation for understanding how Npn-1 functions as a receptor for distinct classes of ligands in vivo. A complex but ordered series of axon guidance decisions during development is critical for the establishment of nervous system structure and function (1Tessier-Lavigne M. Goodman C.S. Science. 1996; 274: 1123-1133Crossref PubMed Scopus (2706) Google Scholar). The vertebrate vascular network is similarly complex, with interconnecting conduits that extend throughout the body that are often in close anatomical proximity to nerve pathways (2Shima D.T. Mailhos C. Curr. Opin. Genet. Dev. 2000; 10: 536-542Crossref PubMed Scopus (77) Google Scholar). Recent evidence suggests that at least some of the same ligand-receptor systems coordinate development of both the nervous system and the cardiovascular system. For example, Eph receptors and their ligands, the ephrins, were first characterized as mediators of repulsive guidance events crucial for correct navigation of neuronal growth cones and migrating neural crest cells (3Holder N. Klein R. Development. 1999; 126: 2033-2044Crossref PubMed Google Scholar, 4Wilkinson D.G. Int. Rev. Cytol. 2000; 196: 177-244Crossref PubMed Google Scholar). Their unexpected role in blood vessel formation was revealed when mutant mice that lacked either ephrin B2 or its cognate receptor EphB4 were shown to die during embryogenesis due to cardiovascular dysfunction (5Gerety S.S. Wang H.U. Chen Z.F. Anderson D.J. Mol. Cell. 1999; 4: 403-414Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar, 6Wang H.U. Chen Z.F. Anderson D.J. Cell. 1998; 93: 741-753Abstract Full Text Full Text PDF PubMed Scopus (1387) Google Scholar). Consistent with this observation, ephrin B2 and EphB4 were shown to have a reciprocal expression patterns in arterial and venous endothelial cells (6Wang H.U. Chen Z.F. Anderson D.J. Cell. 1998; 93: 741-753Abstract Full Text Full Text PDF PubMed Scopus (1387) Google Scholar). Another example of a cell surface receptor whose function is required for development of both the cardiovascular and nervous systems is neuropilin-1 (Npn-1). 1The abbreviations used are: Npn-1neuropilin-1VEGFvascular endothelial growth factorSema3Asemaphorin 3AIP bufferimmunoprecipitation bufferBSAbovine serum albuminAPalkaline phosphatase Npn-1 was first identified by a monoclonal antibody (called A5) isolated in a screen for cell surface proteins capable of mediating axon guidance decisions during neural development (7Takagi S. Tsuji T. Amagai T. Takamatsu T. Fujisawa H. Dev. Biol. 1987; 122: 90-100Crossref PubMed Scopus (141) Google Scholar, 8Takagi S. Hirata T. Agata K. Mochii M. Eguchi G. Fujisawa H. Neuron. 1991; 7: 295-307Abstract Full Text PDF PubMed Scopus (188) Google Scholar). Since its original characterization, molecular, cellular, genetic, and biochemical analyses have shown that Npn-1 is a multifunctional 130-kDa transmembrane protein capable of binding to distinct ligands belonging to completely unrelated protein families: the semaphorins and VEGFs (9He Z. Tessier-Lavigne M. Cell. 1997; 90: 739-751Abstract Full Text Full Text PDF PubMed Scopus (973) Google Scholar, 10Kolodkin A.L. Levengood D.V. Rowe E.G. Tai U.-T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar, 11Soker S. Takashima S. Miao H.-Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2088) Google Scholar). Npn-1 can also serve as a heterophilic cell adhesion molecule in vitro (12Shimizu M. Murakami Y. Suto F. Fujisawa H. J. Cell Biol. 2000; 148: 1283-1293Crossref PubMed Scopus (98) Google Scholar, 13Takagi S. Kasuya Y. Shimizu M. Matsuura T. Tsuboi M. Kawakami A. Fujisawa H. Dev. Biol. 1995; 170: 207-222Crossref PubMed Scopus (175) Google Scholar). neuropilin-1 vascular endothelial growth factor semaphorin 3A immunoprecipitation buffer bovine serum albumin alkaline phosphatase The semaphorins are a large family of proteins that function in axon guidance and cell migration. Class 3 semaphorins, which include the protein semaphorin 3A (Sema3A), are well characterized members of the semaphorin family. In vitro studies have demonstrated that Npn-1 is the ligand binding component of the Sema3A holoreceptor complex (9He Z. Tessier-Lavigne M. Cell. 1997; 90: 739-751Abstract Full Text Full Text PDF PubMed Scopus (973) Google Scholar, 10Kolodkin A.L. Levengood D.V. Rowe E.G. Tai U.-T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar). Npn-1 is expressed in specific classes of developing neurons and is required for repulsive axon guidance mediated by Sema3A (7Takagi S. Tsuji T. Amagai T. Takamatsu T. Fujisawa H. Dev. Biol. 1987; 122: 90-100Crossref PubMed Scopus (141) Google Scholar, 8Takagi S. Hirata T. Agata K. Mochii M. Eguchi G. Fujisawa H. Neuron. 1991; 7: 295-307Abstract Full Text PDF PubMed Scopus (188) Google Scholar, 9He Z. Tessier-Lavigne M. Cell. 1997; 90: 739-751Abstract Full Text Full Text PDF PubMed Scopus (973) Google Scholar, 10Kolodkin A.L. Levengood D.V. Rowe E.G. Tai U.-T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar, 13Takagi S. Kasuya Y. Shimizu M. Matsuura T. Tsuboi M. Kawakami A. Fujisawa H. Dev. Biol. 1995; 170: 207-222Crossref PubMed Scopus (175) Google Scholar, 14Kawakami A. Kitsukawa T. Takagi S. Fujisawa H. J. Neurobiol. 1996; 29: 1-17Crossref PubMed Scopus (247) Google Scholar). Recently, members of the plexin family of large multidomain transmembrane proteins have been shown to physically associate with Npn-1 and together form the functional Sema3A receptor (15Takahashi T. Fournier A. Nakamura F. Wang L.-H. Murakami Y. Kalb R.G. Fujisawa H. Strittmatter S.M. Cell. 1999; 99: 59-69Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar, 16Tamagnone L. Artigiani S. Chen H., He, Z. Ming G.-L. Song H.-J. Chedotal A. Winberg M.L. Goodman C.S. Poo M.-M. Tessier-Lavigne M. Comoglio P.M. Cell. 1999; 99: 71-80Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar). The Npn-1-plexin A1 complex exhibits an enhanced binding affinity for Sema3A as compared with Npn-1 alone (15Takahashi T. Fournier A. Nakamura F. Wang L.-H. Murakami Y. Kalb R.G. Fujisawa H. Strittmatter S.M. Cell. 1999; 99: 59-69Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar), and a mouse with a targeted deletion of one plexin, PlexA3, exhibits defects in class 3 semaphorin-mediated axon repulsion (17Cheng H.J. Bagri A. Yaron A. Stein E. Pleasure S.J. Tessier-Lavigne M. Neuron. 2001; 32: 249-263Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). VEGF family members are critical regulators of vasculogenesis, angiogenesis, and vascular remodeling. The biological effects of VEGFs are mediated by the receptor tyrosine kinases Flt-1 (VEGFR-1), Flk-1/KDR (VEGFR-2), and VEGFR-3 (18Ferrara N. Am. J. Physiol. 2001; 280: C1358-C1366Crossref PubMed Google Scholar, 19Neufeld G. Cohen T. Gengrinovitch S. Poltorak Z. FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3164) Google Scholar). Interestingly, select isoforms of the VEGF family, including VEGF165, also bind with high affinity to Npn-1 (11Soker S. Takashima S. Miao H.-Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2088) Google Scholar). Expression of Npn-1 in endothelial cells enhances both affinity labeling of VEGF165 to VEGFR-2 and VEGF165-induced endothelial cell chemotaxis (11Soker S. Takashima S. Miao H.-Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2088) Google Scholar, 20Miao H.-Q. Soker S. Feiner L. Alonso J.L. Raper J.A. Klagsbrun M. J. Cell Biol. 1999; 146: 233-241Crossref PubMed Scopus (437) Google Scholar). Thus, Npn-1 acts as a VEGF165 co-receptor that augments the ability of VEGF165 to activate VEGFR-2. Npn-1 has also been shown to form a complex with VEGFR-2 (21Whitaker G.B. Limberg B.J. Rosenbaum J.S. J. Biol. Chem. 2001; 276: 25520-25531Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Therefore, Npn-1 serves as a ligand-binding subunit in receptor complexes for structurally distinct ligands, the secreted semaphorins and VEGF165. Consistent with that idea, npn-1 mutant mice exhibit defects in projections of spinal and cranial nerves, and they die during midgestation due to severe cardiovascular dysfunction (22Kawasaki T. Kitsukawa T. Bekku Y. Matsuda Y. Sanbo M. Yagi T. Fujisawa H. Development. 1999; 126: 4895-4902Crossref PubMed Google Scholar, 23Kitsukawa T. Shimizu M. Sanbo M. Hirata T. Taniguchi M. Bekku Y. Yagi T. Fujisawa H. Neuron. 1997; 19: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (560) Google Scholar). Defects observed in these mice could result from a loss of semaphorin-Npn-1 and/or VEGF-Npn-1 signaling. To dissect the molecular mechanisms that contribute to vascular and neuronal functions of Npn-1, we and others have sought to identify structural features of Npn-1 that confer binding to its ligands. Npn-1 has a large extracellular domain, a single transmembrane domain, and a very short cytoplasmic domain (24Fujisawa H. Kitsukawa T. Kawakami A. Takagi S. Shimizu M. Hirata T. Cell Tissue Res. 1997; 290: 465-470Crossref PubMed Scopus (79) Google Scholar). The Npn-1 extracellular domain contains two domains with homology to complement components C1r and C1s (CUB domains, also called “a1” and “a2”) at the amino terminus, followed by two coagulation factor V/VIII domains (CF V/VIII, also called “b1” and “b2”), and one C-terminal MAM (meprin, A5, μ-phosphatase) domain (also called “c”). The secreted class 3 semaphorins contain three domains: a long amino-terminal semaphorin domain (sema), which is the signature domain of this family, an immunoglobulin (Ig) domain, and a positively charged carboxyl-terminal basic domain (25Semaphorin Nomenclature CommitteeCell. 1999; 97: 551-552Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Previous work has shown that the sema domain of Sema3A can bind to Npn-1 and that it is necessary for Sema3A repulsive activity on neurons. The Sema3A Ig-basic region alone cannot elicit biological activity, although it can bind to Npn-1 with a much lower affinity than the intact Sema3A protein (26Nakamura F. Kalb R.G. Strittmatter S.M. J. Neurobiol. 2000; 44: 219-229Crossref PubMed Scopus (248) Google Scholar, 27Raper J.A. Curr. Opin. Neurobiol. 2000; 10: 88-94Crossref PubMed Scopus (405) Google Scholar). Each of the Npn-1 extracellular domains is required for biological activity mediated by Sema3A (28Chen H., He, Z. Bagri A. Tessier-Lavigne M. Neuron. 1998; 21: 1283-1290Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 29Nakamura F. Tanaka M. Takahashi T. Kalb R.G. Strittmatter S.M. Neuron. 1998; 21: 1093-1100Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 30Renzi M.J. Feiner L. Koppel A.M. Raper J.A. J. Neurosci. 1999; 19: 7870-7880Crossref PubMed Google Scholar), but only the CUB domains are required for binding to the sema domain of Sema3A. (31Giger R.J. Urquhart E.R. Gillespie S.K.H. Levengood D.V. Ginty D.D. Kolodkin A.L. Neuron. 1998; 21: 1079-1092Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Moreover, a Npn-1 variant lacking both a1 and a2 CUB domains is incapable of binding to Sema3A but does bind to VEGF165. In contrast, deletion of both Npn-1 b domains abolishes binding of both Sema3A and VEGF165 (31Giger R.J. Urquhart E.R. Gillespie S.K.H. Levengood D.V. Ginty D.D. Kolodkin A.L. Neuron. 1998; 21: 1079-1092Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). These results suggest that the Npn-1 binding determinants for Sema3A and VEGF165 may be distinct. Here we report the identification of amino acid residues within the Npn-1 CUB domains that are required for Sema3A binding but not VEGF165 binding. We find that amino acid substitutions within two adjacent hydrophilic loops of the amino-terminal Npn-1 CUB domain dramatically reduce binding to class 3 semaphorins without affecting VEGF165 binding. In addition to providing insight into the nature of the interactions between Npn-1 and its distinct ligands, these results establish a foundation for understanding how Npn-1 functions as a receptor for distinct classes of ligands in vivo and may also provide a basis for rational drug design. Npn-1 deletion constructs for ligand-binding assays were created by inverse PCR using the ExSite kit (Stratagene). Full-length rat Npn-1 in the expression vector pMT21 served as a template for PCRs using oligonucleotide pairs that flanked the region of interest, including a1, a2, b1, and b2. The oligonucleotides contained a SalI restriction site at their 5′-ends. Following amplification, inverse PCR products were digested with SalI and circularized by ligation yielding Npn-1 deletion constructs: Δ a1+7 (T30–145E), Δ a2 (146C-274K), Δ b1(275C-370P), and Δ b2 (371C-587V). All cloning sites were sequenced, and the expression of correctly sized Npn-1 deletion mutants was confirmed by immunoblotting. Npn-1 mutation constructs including Npn-12I, Npn-12AB, Npn-12C, Npn-13D, and Npn-12ABC were created by three PCR steps. Full-length Npn-1 in the expression vector pMT21 served as a template for the first two PCRs using two pairs of primers. The 5′ primer from the first pair (nucleotides 6–18) includes a native EcoRI site. The 3′ primer from the first pair and the 5′ primer from the second pair overlap and include the introduced mutations. The 3′ primer from the second pair of primers (nucleotides 2515–2539) contains a native EcoRV restriction site. The mixture of these first two purified PCR products then served as a template for the third PCR, using the 5′ primer from the first pair and the 3′ primer from the second pair. Following amplification, the third PCR product was double-digested with EcoRI/EcoRV and subsequently used to replace the corresponding region of wild type Npn-1 in pMT21. This yielded the following Npn-1 mutation constructs: Npn-12I, Npn-12AB, Npn-12C, and Npn-13D. Mutant Npn-12ABC was created by using Npn-12AB as a template for the first two PCRs, and the same primers were used for generating Npn-12C. All PCR products and cloning sites were sequenced, and the expression of correctly sized Npn-1 mutants was confirmed by immunoblotting. AP-tagged ligands were produced in HEK 293T cells. DNA was introduced into cells by LipofectAMINE 2000 (Invitrogen). Conditioned medium was harvested 48 h posttransfection. Ligand concentration was determined as described (37Flanagan J.G. Leder P. Cell. 1990; 63: 185-194Abstract Full Text PDF PubMed Scopus (633) Google Scholar), assuming a specific activity of 2000 units/mg. AP-VEGF cDNA was obtained from Dr. Michael Klagsbrun (Harvard Medical School). VEGF165 used for VEGFR-2 phosphorylation experiments was obtained from Sigma. AP-ligand binding assays were performed as described (31Giger R.J. Urquhart E.R. Gillespie S.K.H. Levengood D.V. Ginty D.D. Kolodkin A.L. Neuron. 1998; 21: 1079-1092Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Briefly, Npn-1 constructs were expressed in COS-1 cells following transfection using LipofectAMINE (Invitrogen). Forty-eight hours posttransfection, cells were incubated with various AP-tagged ligands or with anti-Npn-1 (anti-b2/c domains) (10Kolodkin A.L. Levengood D.V. Rowe E.G. Tai U.-T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar) for cell surface expression. Bound AP activity values were normalized for protein concentration and Npn-1 cell surface expression levels. The normalized AP values for Npn-1 variants were then reported as percentage binding relative to wild-type Npn-1. HEK 293T cells were co-transfected with an expression vector encoding Myc-Plex A1 (4 μg; a gift from Dr. Stephen M. Strittmatter, Yale University) and an expression vector encoding various Npn-1 variants (4 μg) using LipofectAMINE (Invitrogen). After 2 days, cells were lysed with ice-cold immunoprecipitation buffer (IP buffer; 50 mmTris-HCl (pH 8.0), 1% Nonidet P-40, 2 mm EDTA, 0.5 mm polyvinylidene difluoride, 0.3 μmaprotinin, and 10 μm leupeptin). Insoluble proteins were removed by centrifugation at 10,000 × g for 10 min at 4 °C. The concentration of total soluble proteins was determined by the Bradford protein assay (Bio-Rad). Immunoprecipitations were performed using 0.5 mg of protein in a volume of 0.5 ml to which 2 μl of anti-Myc (9E10) ascites was added. Immunocomplexes were recovered using an excess of protein G-Sepharose (Gammanbind; AmershamBiosciences). Following several washes in IP buffer, proteins were eluted in Laemmli sample buffer and then heated at 100 °C for 5 min. Proteins were electrophoresed through standard SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Membranes were then blocked with TBS containing 0.5% nonfat dried milk and 0.1% Tween 20 and probed with anti-Npn-1 (10Kolodkin A.L. Levengood D.V. Rowe E.G. Tai U.-T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar) polyclonal rabbit IgG (0.5 μg/ml). Then blots were probed with peroxidase-conjugated donkey anti-rabbit Ig (Amersham Pharmacia Biotech) (1:5000), and bound secondary antibody was detected with a chemiluminescent peroxidase substrate (Super Signal; Pierce). The same blot was stripped with stripping buffer (62.5 mm Tris base, 2% SDS, and 0.7% β-mercaptoethanol) for 1 h at room temperature and reprobed with anti-Myc primary antibody (1:2000 dilution) and peroxidase-conjugated sheep anti-mouse (Amersham Biosciences) secondary antibody. Control lysates from matched samples were prepared without immunoprecipitation and processed identically as the immunoprecipitated samples described above using anti-Npn-1. HEK 293T cells were transfected with expression vector for VEGFR-2 (4 μg; a gift from Dr. Cam Patterson, University of North Carolina), together with an expression vector for various Npn-1 constructs (4 μg) by the LipofectAMINE method (Invitrogen). After 48 h, the cells were rinsed with Dulbecco's modified Eagle's medium, serum-starved (2–3 h, 37 °C), and then treated with 1 nm VEGF for 5 min at 37 °C. Cells were lysed with IP buffer containing sodium orthovanadate (1 mm), and the samples were immunoprecipitated with anti-Npn-1 (1 μg/ml). Samples were then washed with IP buffer, resolved by SDS-PAGE, and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with TBS containing 3% BSA, 1% normal donkey serum, and 0.1% Tween 20. The membranes were then probed with primary antibody 4G10 anti-phosphotyrosine (0.5 μg/ml; Upstate Biotechnology Inc., Lake Placid, NY) and peroxidase-conjugated sheep anti-mouse (AmershamBiosciences) secondary antibody. Membranes were stripped and reprocessed for VEGFR-2 normalization using the polyclonal Ab R2.2 (21Whitaker G.B. Limberg B.J. Rosenbaum J.S. J. Biol. Chem. 2001; 276: 25520-25531Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). COS-1 cells were transfected with 1 μg of pMT21, pMT21-Npn-1, or pMT21-Npn-12ABC using LipofectAMINE. After 48 h, cells were subjected either to cell surface binding analysis (37Flanagan J.G. Leder P. Cell. 1990; 63: 185-194Abstract Full Text PDF PubMed Scopus (633) Google Scholar) or to cell surface Npn-1 immunocytochemistry. Immunocytochemistry was performed without fixation by incubating cells with blocking solution (5% goat serum in Dulbecco's modified Eagle's medium) for 20 min at 4 °C, followed by anti-Npn-1 (1:250 dilution in Dulbecco's modified Eagle's medium containing 2% goat serum) for 30 min at room temperature. Afterward, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. The cells were washed with PBS an additional three times and incubated with secondary Oregon Green 488 goat anti-rabbit (Molecular Probes, Inc., Eugene, OR) at a dilution of 1:1000. Cells were then visualized under fluorescent microscopy. The iodination of VEGF165 was performed as previously reported (21Whitaker G.B. Limberg B.J. Rosenbaum J.S. J. Biol. Chem. 2001; 276: 25520-25531Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Briefly, 5 μg of carrier-free human recombinant VEGF165 was suspended in 90 μl of Dulbecco's phosphate-buffered saline. To the reaction tube, 1 mCi of Na125I was added, followed by 40 μl of chloramine T (1 μg/μl in 0.5 m sodium phosphate buffer, pH 7.5) and incubated for 1 min. 50 μl of sodium metabisulfite (2 μg/μl in sodium phosphate buffer, pH 7.5) was added to stop the reaction. 500 μl of column elution buffer (0.5% BSA, 0.01% Tween 20 in Dulbecco's phosphate-buffered saline) was added to the reaction and transferred to preequilibrated PD-10 column for separation from unreacted iodine. The specific activity was corrected for column recovery and varied from 5,500 to 7,500 Ci/mmol. The saturation analysis was performed as previously reported (21Whitaker G.B. Limberg B.J. Rosenbaum J.S. J. Biol. Chem. 2001; 276: 25520-25531Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Briefly, COS-1 cells transiently expressing either the Npn-1 or the Npn-12ABC mutant receptor were plated at 2 × 105 cells/well in a 12-well plate 24 h prior to experimentation. The following morning, the cells were rinsed with 1 ml of binding buffer (Dulbecco's modified Eagle's medium, 0.2% BSA, 25 mm HEPES) and were preequilibrated in the same buffer for 1 h at 4 °C. Increasing concentrations (30–5000 pm) of [125I]VEGF165 were added in binding buffer containing a protease inhibitor mixture (final concentration as follows: leupeptin, 10 μg/ml; antipain, 10 μg/ml; aprotinin, 50 μg/ml; benzamine, 100 μg/ml; soybean trypsin inhibitor, 100 μg/ml; bestatin, 10 μg/ml; pepstatin 10 μg/ml; phenylmethylsulfonyl fluoride, 0.3 mm) and 1 μg/ml heparin in the presence or absence of 30 nm unlabeled VEGF165 to estimate nonspecific binding. The nonspecific binding was linear over the indicated tracer concentration range (data not shown). The binding reaction was allowed to reach equilibrium (4 h at 4 °C), and the unbound ligand was removed by washing three times (1 ml) with ice-cold BSA-free binding buffer. The cells were lysed with 250 μl of RIPA buffer (20 mm Tris-HCl, pH 7.4, 100 mm NaCl, 1 mm EDTA, 10 mm NaI, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 1% BSA, 0.1% SDS) and counted using a γ-counter. The maximum number of binding sites (Bmax) and the equilibrium dissociation constant (Kd) values were obtained using the Prism software package, which performs a statistical assessment of goodness of fit to a one-binding site versus a two-binding site model. This methodology is preferred over the Scatchard analysis, since it does not require a transformation and linearization of the data, which is known to distort the experimental errors associated with radioligand-binding data (38Motulsky H.J. Ransnas L.A. FASEB J. 1987; 1: 365-374Crossref PubMed Scopus (1106) Google Scholar). In all cases described herein, the optimal fit of the data was to a one-binding site model (data not shown). The data points in all curves were determined in triplicate. The lower Bmax value observed for the mutant receptor (as compared with the wild type receptor; see Table I) can be attributed to the lower expression level of mature protein for the mutant receptor in the COS-1 transient expression system (data not shown). The bovine spermadhesin CUB domain was displayed, and side chain was changed to the corresponding residues in Npn-1 using the program O (39Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). Fractional solvent accessibilities were calculated using the program X-PLOR (40Brunger A.T. X-PLOR, Version 3.1., a system for x-ray crystallography and NMR. Yale University Press, New Haven, CT1992Google Scholar). We set out to identify structural features of Npn-1 that confer binding to VEGF165 and Sema3A, two structurally unrelated ligands with distinct functions. Using a PCR-based mutagenesis approach, we constructed a battery of Npn-1 variants harboring different deletions within the CUB and CF-V/VIII domains. Npn-1 variants lacking individual CUB (a1 or a2) or CF-V/VIII (b1 or b2) domains were generated (Fig. 1 A) and expressed in COS-1 cells. Expression of these variants was confirmed by anti-Npn-1 Western blots and cell surface immunostaining in the absence of detergent. Each variant was tested for its ability to bind to Sema3A and VEGF165 ligands fused at their N termini to alkaline phosphatase (AP-Sema3A and AP-VEGF165). Both of these AP fusion proteins bind with high affinity to Npn-1 (10Kolodkin A.L. Levengood D.V. Rowe E.G. Tai U.-T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar, 20Miao H.-Q. Soker S. Feiner L. Alonso J.L. Raper J.A. Klagsbrun M. J. Cell Biol. 1999; 146: 233-241Crossref PubMed Scopus (437) Google Scholar). Fig. 1depicts four of the Npn-1 deletion variants and their AP-Sema3A and AP-VEGF165 binding properties. Deletion of either the a1 or a2 CUB domains abolished AP-Sema3A binding but had relatively little effect on VEGF165 binding (Fig. 1, C and D). In contrast, deletion of the b1 CF-V/VIII domain abolished binding to both ligands (Fig. 1, C and D). Deletion of the b2 CF-V/VIII domain did not abolish binding to either ligand (Fig. 1, C and D), although reduced binding of AP-VEGF165 to the b2 deletion variant was consistently observed. These results suggest that each of the CUB domains is essential for Sema3A but not VEGF165binding, and the b1 CF-V/VIII region is required for binding to both ligands. To further refine our characterization of distinct ligand binding sites on Npn-1, candidate Sema3A-binding residues were sought by generating a three-dimensional model of the Npn-1 a1 CUB domain. Using the crystal structure of the bovine spermadhesin CUB domain (32Romero A. Romao M.J. Varela P.F. Kolln I. Dias J.M. Carvalho A.L. Sanz L. Topfer-Petersen E. Calvete J.J. Nat. Struct. Biol. 1997; 4: 783-788Crossref PubMed Scopus (129) Google Scholar) and an alignment of the Npn-1 a1 and spermadhesin CUB domain amino acid sequences, we identified four loop regions in the Npn-1 a1 CUB domain that are likely to be solvent-exposed (Fig. 2, labeled red (2I),pink (2AB), light green (2C), and dark green (3D)). Three of these (2AB, 2C, and 3D) reside on one side of the predicted a1 CUB domain, whereas the fourth (2I) lies on the opposite side. Solvent-exposed regions were sought because they are likely to mediate interactions with ligands, and mutations at such sites are less likely to disrupt global structure. Residues of the four regions selected for mutation (2A, 2B, 2C, and 2I) were not only solvent-exposed but