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Published as doi: 10.1096/fj.05-5504fje.
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(The FASEB Journal. 2006;20:1513-1515.)
© 2006 FASEB

C terminus of RGS-GAIP-interacting protein conveys neuropilin-1-mediated signaling during angiogenesis

Ling Wang*,{dagger}, Debabrata Mukhopadhyay{dagger},1,2 and Xiaolei Xu*,1,2

* Department of Biochemistry and Molecular Biology,

{dagger} Mayo Clinic Cancer Center, Mayo Clinic College of Medicine, Rochester, Minnesota, USA

1Correspondence: D. M., Department Biochemistry and Molecular Biology, Gugg 1401A, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905. E-mail: mukhopadhyay.debabrata{at}mayo.edu; X. X., Department Biochemistry and Molecular Biology, Gugg 1701C, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905, USA. E-mail: xu.xiaolei{at}mayo.edu

ABSTRACT

Initially, it was thought that there was no intracellular signaling mediated by NRP-1 alone in response to its ligands. However, the emerging data from our group as well as others suggest that the signaling through NRP-1 actually promotes angiogenesis and is mediated through its C-terminal domain and downstream molecules such as phosphoinositide 3-kinase. Hence, understanding the signal transduction pathways mediated by NRP-1 and identification of its downstream molecules are of importance. By using both in vivo zebrafish model and in vitro tissue culture system, we have shown that the C-terminal three amino acids of NRP-1 (SEA-COOH) are required for NRP-1-mediated angiogenesis. Furthermore, knocking down of RGS-GAIP-interacting protein C terminus (GIPC) in zebrafish, which is associated with C-terminal domain of NRP-1, exhibits similar vasculature phenotypes to those from NRP-1 null. Specific and effective silencing of GIPC in vascular endothelium results in inhibition of NRP-1-mediated migration. In both cases as described, PDZ domain of GIPC is responsible for its function. Taken together, our data suggest a novel role of GIPC in angiogenesis and vessel formation and also support our hypothesis that NRP-1 can facilitate downstream signaling to promote angiogenesis through GIPC.—Wang, L., Mukhopadhyay, D., Xu, X. C terminus of RGS-GAIP-interacting protein conveys neuropilin-1-mediated signaling during angiogenesis.


Key Words: NRP-1 • VPF/VEGF • endothelial cells • zebrafish • PDZ-domain

THE ANGIOGENIC PROCESS is critically regulated by the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), which acts through binding to its receptors VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and neuropilin-1 (NRP-1; ref 1 2 3 4 ). Gene-targeting studies have documented embryonic lethality in NRP-1 null mice (5) . Morpholino-mediated knockdown of NRP-1 in zebrafish embryos resulted in vascular defects, most notably impaired circulation in the intersegmental vessels (6 7 8) . Although these in vivo findings have demonstrated a critical role of NRP-1 in vascular development, the involved molecular signaling events of NRP-1 in response to VPF/VEGF are still unclear. Although the original dogma declared NRP-1 only as a coreceptor of VEGFR-2 in VPF/VEGF signaling (9 10 11) , recent studies have indicated that NRP-1 promotes VPF/VEGF-induced migration (12) and adhesion (13) in endothelium independent of VEGFR-2. Therefore, it is tempting to explore distinct pathways for NRP-1-mediated VPF/VEGF signaling in endothelial cells (ECs).

NRP-1 is a 130–135 kDa cell surface glycoprotein. It contains a relatively large extracellular domain of 860 amino acids, a very short transmembrane domain of 23 amino acids, and an intracellular domain of 39 amino acids (9) . Semaphore function in neuronal cells seems independent on the intracellular domain of NRP-1 (2 , 14) . However, the transmembrane and intracellular domains of NRP-1 share >90% amino acid identity across species (15 ,16) , suggesting an important role for these domains in terms of the NRP-1 functions. By generating the chimeric mutant receptor EGNP-1{Delta}SEA, in which the C-terminal three amino acids of NRP-1 (SEA-COOH) were deleted, we have shown that the C-terminal three amino acids (SEA-COOH) are essential for NRP-1-mediated human umbilical vein endothelial cells (HUVECs) migration (12) .

These observations of the importance of the NRP-1 intracellular domain, especially the C-terminal three amino acids (SEA-COOH), in cell function have led to a search for their binding partners. A recent study has shown that the C-terminal three amino acids of NRP-1 (SEA-COOH), which are conserved from Xenopus to human (15 , 16) , are required for the interaction with the C-terminal two-thirds of neuropilin-1 interacting protein (NIP), a PDZ domain-containing protein, in the dynamic axon growth cone (17) . Possible functions of PDZ domain-containing proteins are to act as molecular adapters that target proteins to proper subcellular compartments and to assemble signal transduction components into closely associated protein complexes (18 19 20) . NIP has also been independently cloned as RGS-GAIP-interacting protein (GIPC), where it was identified by virtue of its interaction with the C terminus of RGS-GAIP (a G{alpha}i3-associated protein located to the membrane of clathrin-coated vesicles) and was suggested to participate in the regulation of clathrin-coated vesicular trafficking by association with the G-protein-coupled signaling complex (21) .

Prompted by the above investigations, we hypothesized that the C-terminal three amino acids (SEA-COOH) of NRP-1 mediate VPF/VEGF functions by directly binding PDZ domain-containing GIPC, which provides a link between NRP-1 and its downstream signaling molecules. In this study, we have tested this hypothesis by using both a vascular-specific transgenic zebrafish line [TG(fli1:egfp)] and tissue culture system. Zebrafish is being recognized as an excellent in vivo model for analyzing genes that regulate vascular development, partially due to the conservation of molecular mechanisms and morphological structures of vessel growth among vertebrate species (22 23 24 25 26) . Zebrafish intersegmental vessels correspond to mammalian capillary sprouts, whereas the axial vessels correspond to major blood vessels, such as arteries (6) . The transparency of the embryos and the rapid ex utero development of a circulatory system (27) permit direct visual analysis in a living animal. The process of angiogenesis in zebrafish has been well documented (28) . To explore the functions of NRP-1 that are independent of VEGFR-1 and VEGFR-2 in transducing VPF/VEGF signals in ECs, we have established an approach to engineer a series of chimeric constructs, where the extracellular domains of each receptors were replaced by the extracellular domain of epidermal growth factor (EGF) receptor (EGFR; ref 29 ). We have demonstrated the feasibility of the approach by showing that HUVECs did not express the EGFR and did not respond to EGF under the conditions of our experiments (80% confluence; ref 29 ). We also demonstrated the reliability of this approach by showing that the intracellular domains of these chimeric proteins had the same cellular activities as the wild-type (WT) proteins (29) .

These studies confirmed that the C-terminal three amino acids of NRP-1 are the key functional domains for NRP-1-mediated angiogenesis. Furthermore, we identified and characterized a NRP-1-interacting protein in endothelium, GIPC, which is required for NRP-1-mediated VPF/VEGF induced angiogenesis. By interacting with the C-terminal domain of the NRP-1 (SEA-COOH), GIPC conveys the signal of the VPF/VEGF to regulate the migration of the ECs during the process of angiogenesis.

MATERIALS AND METHODS

Zebrafish lines and maintenance
Zebrafish were maintained and bred as described previously (30) . All experiments were performed by using the TG(fli1:egfp)y1 l transgenic line that has been described previously (31) . Embryos were staged according to Kimmel et al. (32) .

Bioinformatics analysis
Bioinformatics analysis is performed to analyze the zebrafish homologue of GIPC (GenBank accession no.: AY690668). Clustal W is used to demonstrate the conserved domains between zebrafish and human GIPC orthologues.

Whole-mount in situ hybridization and histology
Whole-mount in situ hybridization was performed essentially as described by Thisse et al. (33) . In situ probes were generated from polymerase chain reaction (PCR) products using T7 promoter tagged primer pairs. Whole-mount zebrafish in situ hybridization was carried out using digoxigenin-labeled antisense riboprobes, which contain digoxigenin-11-uridine triphosphate (Roche) that is visualized using the BM purple alkaline phosphatase substrate (Roche). Embryos were cleared and flat-mounted in the magic solution for photography. For histological analysis, specimens were fixed in 4% paraformaldehyde, dehydrated, and embedded in a plastic medium (JB-4) and sectioned.

Morpholinos
Morpholinos were designed targeting splice donors of the targeted genes to efficiently block premRNA splicing. Morpholinos against the following genes were designed: the exon 5-intron 5 boundary sequence of NRP-1a, the exon 3-intron 3 boundary sequence of NRP-1b, and the exon 3-intron 3 boundary sequence of GIPC. We purchased Morpholino antisense oligonucleotides from Gene Tools. The injection solution was prepared as described previously (34) . The sequences are available on request. Morpholino solutions with 1.25 and 10 ng morpholinos were injected into zebrafish embryos at the 1–4 cell stage. Embryos were examined for vascular defects at 48 h postfertilization (hpf). Quantitative RT-PCR (Ambion) was used to monitor the splicing events. All experiments were repeated at least three times.

mRNA microinjection
The human full-length NRP-1 and a construct with last three amino acids deletion (SEA), the human full-length GIPC, and its PDZ domain deletion mutant were cloned into the pCS2 vector, respectively. The transcribed mRNAs were injected together with the NRP-1 morpholino or alone into 1–4 cell staged embryos. With the use of a splicing donor morpholino, it is possible to reduce the endogenous expression of the gene, without disturbing the expression of ectopic mRNAs. All experiments were repeated at least three times.

Imaging
Imaging of blood vessels in TG(fli1:egfp)y1 embryos was performed using a Zeiss confocal laser microscope as described previously (31) . Transmitted light images were obtained with a Leica MZ FLIII microscope equipped with a Nikon COOLPIX8700 digital camera.

Cell culture
HUVECs (Clonetics, San Diego, CA) were cultured as described previously (12) . HUVECs were grown on 30 µg/ml vitrogen-coated dishes in EGM-MV bullet kit (5% FBS in EBM with 12 µg/ml bovine brain extract, 1 µg/ml hydrocortisone, and 1 µg/ml GA-1000). HUVECs (passage 3 or 4) that were {approx}80% confluent were used for most experiments. Cells were serum starved in 0.1% FBS in EBM for 24 h before testing.

Immunoprecipitation and Immunoblotting
HUVECs were lysed with cold immuneprecipitation buffer (50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS), which contains 1 mM PMSF, 1 µg/ml leupeptin, 0.5% aprotinin, and 2 µg/ml pepstatin A; 500 µg of lysate protein were incubated with 1 µg of antibody (Ab) at 4°C for 2 h, and then with 50 µl of protein G-conjugated agarose-beads at 4°C for 2–5 h. After the beads were washed with the same buffer, immunoprecipitates were resuspended in 2x SDS sample buffer for immunoblotting analysis. All experiments were repeated at least three times. Antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

RNA interference
siRNA were purchased from Santa Cruz Biotechnology. For siRNA transfection, HUVECs at 70% confluency were transfected in OPTI-MEM I reduced serum medium medium (Invitrogen, Carlsbad, CA) with the siRNA duplexes using Oligofectamine reagent (Invitrogen). After 4 h, the complete HUVEC medium was added and then the cells were incubated for another 24 h before the functional experiments were performed.

Migration assay
Cell migration was performed as described previously (12) . In brief, serum-starved HUVECs seeded into the upper chamber of the transwell for a 2 h incubation prior to migration to VPF/VEGF or EGF. The migrated, stained cells are counted in a spectrofluorometer (Spectrafluor; TECAN) with Delta Soft 3 software. Data are mean ± SD of quadruplicate values. All experiments were repeated at least three times. Comparison of numerical data was evaluated by Student’s t test. A P value of <0.05 was considered statistically significant.

RESULTS

C-terminal three amino acids of NRP-1 (SEA-COOH) are required for NRP-1-mediated zebrafish angiogenesis
C-terminal three amino acids of NRP-1 (SEA-COOH) are conserved from Xenopus to human, which suggests they might be key functional domains for NRP-1 signaling (16) . Because the in vivo process of angiogenesis in zebrafish has been well documented (28) , we would like to investigate the functions of the C-terminal three amino acids (SEA-COOH) of NRP-1 in zebrafish. Recently, two zebrafish NRP-1 homologues have been identified, indicating that NRP-1 gene duplicates during the evolution of the teleost fish. Both genes have been shown to be required for vascular development (6 7 8) .

To dissect functions of individual domain of NRP-1 in angiogenesis, we first knocked down endogenous functions of zNRP-1a and zNRP-1b during zebrafish embryogenesis using morpholino antisense oligos. Morpholino antisense oligos were injected into TG(fli1:egfp) embryos, since all the vasculature is labeled by the GFP signal, which can be detected by confocal fluorescent microscope in a living fish. Starting at 24 somite (24s) stage, individual angioblast sprouts from aorta to form intersegmental vessles (ISV) that consists of ventral, middle, and dorsal angioblasts. Dorsal angioblasts migrate dorsally between somites to form T-shaped cells, which later develop into two lines of dorsal longitudinal anastomotic vessel (DLAV) that are connected with each other. In addition, a group of angioblasts migrate ventrally from aorta into the yolk to form the subintestinal vein (SIV; ref 28 ). In this study, we found that knockdown of either zNRP-1a or zNRP-1b disrupted the formation of DLAV, SIV, and ISV in a dosage dependent pattern. TG(fli1:egfp) embryos injected with 1.25 ng of either zNRP-1a or zNRP-1b morpholino disrupted the formation of DLAV and SIV (Fig. 1 ), while injection of 10 ng NRP-1a or 1b morpholino resulted in much more severe vessel defects in DLAV, ISV, and SIV and a reduction in body size (Fig. 1) with failure of circulation. Coinjection of morpholinos against both zNRP-1a and zNRP-1b led to similar vessel defects in low dosage (1.25+1.25 ng) or completely ablated the formation of DLAV and SIV in high dosage (10+10 ng; Fig. 1 ), suggesting a functional redundancy of the two NRP-1 homologues. It seems that knockdown NRP-1 in zebrafish disrupts angiogenesis but not the vasculogenesis process, as both the aorta and vein develop normally. These specific phenotypes can be rescued by injection of human NRP-1 mRNA. As shown in Fig. 1 , the embryos coinjected with NRP-1a/NRP-1b morpholinos and hNRP-1 mRNA had normal DLAV and SIV with established blood circulation. These data indicate that the functions of NRP-1 are conserved between zebrafish and human.


Figure 1
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  		Figure 1. NRP-1 is required for DLAV and SIV formation during zebrafish angiogenesis. Shown are confocal images of 2 days postfertilization (dpf TG(fli1:egfp) embryos. Dorsal is up and anterior is left. As indicated in top panel, different amounts of morpholinos against zNRP-1a or/and zNRP-1b were injected into 1–4 cell stage embryos. First upper rank shows defects of DLAV and ISV; second upper rank shows defects of SIV; and lower rank shows the change of morphology. Arrow indicates DLAV, brackets indicate ISV and SIV, respectively; and double arrowheads indicate DA and PCV. Lower panel) Percentage of vessel defects (n=90 and 50 at 1.25 and 10 ng morpholino injection groups, respectively).

To confirm the functional importance of the C-terminal domain of NRP-1, as suggested by our in vitro studies (12) , we injected hNRP-1{Delta}SEA mRNA, which deleted the C-terminal three amino acids (SEA-COOH) in human NRP-1 and analyzed its effects on zebrafish angiogenesis. In contrast to injection of full-length hNRP-1, coinjection of hNRP-1{Delta}SEA mRNA with zNRP-1 morpholinos cannot rescue the vascular-specific phenotypes of NRP-1 knockdown. On the contrary, we observed disastrous vessel defects in the formation of DLAV, ISV, and SIV (Fig. 2 ), lack of blood flow in these vessels, and more significant reduction in body size. These data suggest that hNRP-1{Delta}SEA may function as a dominant negative mutant. Indeed, injection of hNRP-1{Delta}SEA alone disrupted the formation of DLAV, ISV, and SIV, which resembles the phenotypes generated from zNRP-1 knockdown (Fig. 2) . As expected, injection of hNRP-1 mRNA showed normal vessel development.


Figure 2
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  		Figure 2. C-terminal three amino acids (SEA-COOH) of NRP-1 are required for NRP-mediated angiogenesis. Shown are confocal images of 2 dpf TG(fli1:egfp) embryos. Dorsal is up and anterior is left. As indicated in top panel, embryos were injected with combinations of different amounts of morpholinos against zNRP-1a/zNRP-1b and mRNA transcripts of hNRP-1{Delta}SEA or hNRP-1. Upper three ranks reveal phenotypes of morpholinos and mRNA treatment. Arrow indicates DLAV; brackets indicate ISV and SIV (subintestinal vein), respectively; and double arrowheads indicate DA and PCV (posterior cardinal vein). Lower rank is immunoblotting result that reveals protein expression concentration of human hNRP-1{Delta}SEA or hNRP-1 in zebrafish. Lower panel) Percentage of vessel defect (n=60, 30, 85, and 120, at 0.1 ng hNRP-1{Delta}SEA, 0.3 ng hNRP-1{Delta}SEA, 0.1 ng hNRP-1, and 0.3 ng hNRP-1 injection groups, respectively).

In summary, our data suggest that NRP-1 is required for the migration of angioblast to form either DLAV or SIV during zebrafish angiogenesis. This is consistent with our previous data in HUVEC cells that NRP-1 alone mediated the migration of ECs. Our in vivo data further underscored the importance of the C-terminal three amino acids in the function of the NRP-1 as the signaling mediator during angiogenesis, especially during the migration event.

C-terminal domain of NRP-1 interacts with GIPC in ECs
It is expected that the C-terminal domain of NRP-1 couples with intracellular signaling molecules directly or through an adapter protein to convey its functions. As stated before, we hypothesized that NRP-1 transduces the signal in ECs through its C-terminal domain by interacting with GIPC. To test this hypothesis, we initially sought to confirm the protein-protein interaction between NRP-1 and GIPC in HUVECs. We characterized the expression of GIPC in HUVECs by using a polyclonal antibody against GIPC protein. As shown in Fig. 3 A by immunoprecipitation and immunoblotting, anti-GIPC Ab detected a specific band with molecular masses {approx}40 kDa, which corresponds to the size of GIPC found in the nervous system (39 kDa). Meanwhile, coimmunoprecipitation experiment was also performed to confirm the physical interaction between endogenous NRP-1 and GIPC. Cell lysates were immunoprecipitated with an anti-NRP-1 Ab, and then immunoblotting with anti-GIPC Ab was performed. As shown in Fig. 3A , GIPC coimmunoprecipitated with NRP-1 from HUVEC extracts. Furthermore, we investigated the role of the C-terminal three amino acids of NRP-1 (SEA-COOH) as the interacting domain with GIPC. EGNP-1 and EGNP-1{Delta}SEA were transiently expressed in HUVECs. Immunoprecipitation with anti-GIPC Ab and then immunoblotting with anti-NRP-1 Ab were carried out. Anti-goat IgG was used as control in immunoprecipitation. As shown in Fig. 3B , EGNP-1 could interact with GIPC, but deletion of the C-terminal three residues of NRP-1 (EGNP-1{Delta}SEA) abolished its interaction with GIPC, although the expression concentration of the truncated NRP-1 protein was comparable with the full-length construct. These observations confirm the specific interaction mediated by the C-terminal three amino acid (SEA-COOH) between NRP-1 and GIPC in an EC line. In addition, we observed stronger interaction between NRP-1 and EGNP-1 with GIPC, respectively, under stimulation with VEGF or EGF.


Figure 3
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  		Figure 3. GIPC physically interacts with C-terminal domain of NRP-1 in HUVEC cells. Immunoblotting and coimmunoprecipitation of GIPC and NRP-1 were performed. A) GIPC expressed in HUVECs and interacted with endogenous NRP-1. NRP-1 was immunoprecipitated from cell lysate by using anti-NRP-1 Ab, and resulting immunoprecipitates were probed with anti-GIPC-antibody. GIPC was coimmunoprecipitated with NRP-1 that showed the same size band ({approx}40 kDa) with control with anti-GIPC Ab for immunoprecipitation. B) C-terminal three amino acids of NRP-1 (SEA-COOH) mediated interaction of GIPC and NRP-1. GIPC was immunoprecipitated from solubilized HUVECs extract by using anti-GIPC Ab, and the resulting immunoprecipitates were probed with anti-NRP-1-antibody. NRP-1 and EGNP-1 were coimmunoprecipitated with GIPC, respectively, but EGNP-1{Delta}SEA did not show interaction (upper rank). Cellular NRP-1 showed by immunoblotting with anti-NRP-1 Ab (lower rank).

GIPC expresses in zebrafish vasculature and is required for zebrafish angiogenesis
By searching the zebrafish genomic database, we identified genomic sequence of zebrafish NIP (zGIPC). The predicted amino acid of zGIPC is highly homologous (82% identity) to the human GIPC protein sequences (Fig. 4 A). The expression patterns of zGIPC during embryogenesis were characterized by whole mount in situ hybridization at 18s stage, 24 hpf (zebrafish embryonic stages are described by Kimmel et al., 32 ) and 48 hpf. In the embryos of 18s, 24 and 48 hpf, the expression of zGIPC was observed in brain and probably the central nervous system (CNS), which is consistent with the previous finding in mouse. Interestingly, the GIPC transcript was detected in embryo 18s stages, in the trunk region, two bilateral stripes of GIPC-positive cells (Fig. 4B ), which reaches the tailed region and converge toward the ventral midline to merge into a single stripe that terminates at the ventral region of the tail (Fig. 4B ). These cells give rise to the ECs. Furthermore, we detected the expression of zGIPC in developing vessels including trunk vessels [dorsal aorta (DA) and posterior cardinal vein (PCV)] and caudal vein plexus (CVP) at 24 and 48 hpf embryos (Fig. 4B ). The transient expression pattern is consistent with those from NRP-1a and 1b, which has been reported by the other investigators (6 7 8) . This result supports our hypothesis that GIPC plays a role during the angiogenesis, probably by conveying the signals of NRP-1.


Figure 4
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  		Figure 4. Zebrafish GIPC expresses in vessels and involves in angiogenesis. A) Shown is alignment of the deduced amino acid sequences of zebrafish GIPC (zGIPC) with human GIPC (hGIPC). Amino acids that are conserved between zGIPC and hGIPC are shaded. B) Expression of GIPC in zebrafish. Upper rank reveals whole mount in situ hybridization of zGIPC in different stage of zebrafish embryos. Dorsal is up and anterior is left. Lower rank shows transverse section through trunk of different stage of zebrafish embryos. Dorsal is up. Arrowheads depict zGIPC-expressing cells. C) GIPC is required for DLAV and SIV formation during zebrafish angiogenesis, which resembles NRP-1 knockdown. Shown are confocal images of 2 dpf TG(fli1:egfp) embryos. Dorsal is up and anterior is left. Two different amounts of zGIPC morpholinos were injected into 1–4 cell stage embryos. Upper three ranks reveal phenotypes of morpholinos and mRNA treatment. Arrow indicates DLAV (the dorsal longitudinal anastomotic vessel); brackets indicate ISV and SIV, respectively, and double arrowheads indicate DA and PCV. Lower panel) Percentage of vessel defects (n=120 and 65 at 1.25 and 10 ng GIPC morpholion injection groups, respectively).

To analyze the function of zGIPC during zebrafish angiogenesis, we knocked down zGIPC expression using morpholino antisense oligoes. zGIPC morpholino (1.25 and 10 ng) was injected into TG(fli1:egfp) embryos at the 1–4 cell stage. Injection of 1.25 ng zGIPC morpholino into TG(fli1:egfp) embryos disturbs the formation of DLAV and SIV. Injection of 10 ng zGIPC morpholino results in more severe vessel defects including DLAV, ISV, and SIV (Fig. 4C ). Circulation in DLAV and ISV was diminished or blocked. The body size was reduced as well. These phenotypes resemble those from NRP-1 knockdown, which suggests that zGIPC is involved in NRP-1 signaling pathway.

Previous data in neuronal studies suggested that the PDZ domain of GIPC might interact with the C-terminal three amino acids of NRP-1 (SEA-COOH; ref 17 ). To explore the role of PDZ domain of GIPC in angiogenesis, we constructed two plasmids to generate mRNAs for human GIPC (hGIPC) or human GIPC with a PDZ deletion (hGIPC{Delta}PDZ). Coinjection of hGIPC mRNA with zGIPC morpholino rescued the phenotype of zGIPC knockdown and thus displayed normal vessel and circulation (Fig. 5 ). However, coinjection of hGIPC{Delta}PDZ mRNA with zGIPC morpholino did not rescue zGIPC knockdown phenotype, despite similar levels of protein expression (Fig. 5) . Interestingly, microinjection of hGIPC{Delta}PDZ mRNA alone exhibited similar phenotypes of GIPC knockdown, including the defects of DLAV, ISV, and SIV (Fig. 5) , whereas microinjection of hGIPC revealed normal circulation and light vessel overgrowth (Fig. 5) . Taken together, these results suggest that the PDZ domain of GIPC plays an important role in GIPC-mediated NRP-1-induced angiogenesis, probably by interacting with the C-terminal domain of NRP-1 (SEA-COOH).


Figure 5
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  		Figure 5. PDZ domain of GIPC is required for GIPC-mediated angiogenesis. Shown are confocal images of 2 dpf TG(fli1:egfp) embryos. Dorsal is up and anterior is left. As indicated in top panel, embryos were injected with combinations of morpholinos against GIPC morpholino and different amounts of mRNA for hGIPC or hGIPC{Delta}PDZ. Upper three ranks reveal phenotypes of morpholinos and mRNA treatment. Arrow indicates DLAV, brackets indicate ISV and SIV, respectively, and double arrowheads indicate DA and PCV. White arrows point to the overgrowth. Bottom rank shows immunoblotting results that reveal the protein expression concentration of human hGIPC or hGIPC{Delta}PDZ mRNA in zebrafish. Lower panel) Percentage of vessel defects (n=60, 30, 80, and 110, at 0.1 ng hGIPC{Delta}PDZ, 0.3 ng hGIPC{Delta}PDZ, 0.1 ng hGIPC, and 0.3 ng hGIPC injection groups, respectively).

GIPC is involved in NRP-1-mediated EC migration
To address the functions of GIPC during the NRP-1 mediated EC migration, we analyzed the role of GIPC in NRP-1/EGNP-1-mediated HUVECs migration. For this purpose, we first knocked down GIPC in HUVEC cell by using RNA interference-mediated silencing. As shown in Fig. 6 A, GIPC siRNA specifically and effectively silenced the expression of GIPC but not that of NRP-1 or EGNP-1. Migration assay was then carried out in HUVECs transduced with EGNP-1 by using transwell chamber. After 2 h of incubation, EC migration induced by EGF in EGNP-1 transduced HUVEC increased {approx}2-fold (P<0.01 in a Student’s t test), than that of untransfected HUVECs or HUVECs transduced with LacZ (Fig. 6B ). However, the transfection of HUVECs with GIPC siRNA significantly blocked EGNP-1-mediated HUVECs migration (P value is 0.0156, 0.0042, and 0.0014 in Student’s t test, in 0.03, 0.3, and 3 pM GIPC siRNA sliencing groups, respectively) in EGNP-1 transduced HUVEC siRNA (Fig. 6B ), whereas scrambled siRNA did not show any statistical significance (P value is 0.0557 in a Student’s t test). As shown in Fig. 6B , HUVECs transduced with increasing amounts of GIPC siRNA progressively blocked migration of EC. In consistency with our in vivo data, these results further suggest that GIPC is involved in NRP-1-mediated EC migration.


Figure 6
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  		Figure 6. GIPC is required for NRP-1 mediated HUVECs migration. A) Inhibition of GIPC expression by siRNA. HUVECs were transfected with siRNA against GIPC in different concentration and immunoblotting with anti-GIPC Ab were carried out using cellular lysates. Control siRNA was used as control. B) GIPC is involved in NRP-1/EGNP-1-mediated HUVECs migration. Migration assay was performed in HUVECs transfected with EGNP-1 and then transfected with siRNA. Knocking down of GIPC inhibited EGF-stimulated HUVECs migration in a dosage dependent manner. *P < 0.05; **P < 0.01 in a Student’s t test. Immunoblotting was carried out to check expression levels of GIPC in these cells, which is shown in upper part.

DISCUSSION

By using the chimeric receptors of EGNP-1 (fusing the N-terminal domain of EGFR to the transmembrane and intracellular domains of the WT NRP-1) and its mutant EGNP-1-{Delta}SEA [fusing the N-terminal domain of EGFR to the WT transmembrane domain and a mutated C-terminal intracellular domain of the NRP-1 harboring three amino acid (SEA-COOH) deletion], we have found that NRP-1/EGNP-1 mediated HUVEC migration through its intracellular domain. More interestingly, we demonstrated that the C-terminal three amino acids (SEA-COOH) of NRP-1 are essential for this function (12) . On the basis of these findings, we set out to further confirm the functional importance of the C-terminal three amino acids (SEA-COOH) of NRP-1 in an in vivo zebrafish model. Combining evidence from morpholino knockdown and mRNA rescue, we demonstrated that C-terminal three amino acids (SEA-COOH) of NRP-1 are essential for NRP-1-mediated VPF/VEGF-induced angiogenesis.

The fact that the C-terminal three amino acids of NRP-1 (SEA-COOH) are key functional domains in NRP-1 signaling prompted us to search for their binding partners. The domain might be coupled with intracellular signaling molecules directly. The C-terminal three residues of NRP-1 (SEA-COOH) agree with the consensus sequence of the class I postsynaptic density protein, disc-large, zo-1 (PDZ) domain binding motif. The PDZ domain has been found to be a novel modular domain for specific protein-protein interaction (35 36 37 38 39 40 41) , which binds directly to a short consensus sequence in transmembrane proteins, T/S-X-V-COOH (42) . The protein-protein interactions mediated by PDZ-containing proteins leads to diverse biology outcomes (43) , such as muscle contraction (44) , synapse formation (45 , 46) , and vesicular trafficking (21) . By using a yeast two-hybrid screen, Cai and Reed (17) have shown that the C-terminal three amino acids of NRP-1 (SEA-COOH) are responsible for interaction with the PDZ domain-containing C-terminal two-thirds of NRP-1 interacting protein (NIP or GIPC) in developing nervous systems (17) . It is thus possible that GIPC also acts as the molecular adapter to mediate NRP-1-induced signaling in EC. In this study, we demonstrated that both GIPC and NRP-1 express in HUVECs. We revealed the interaction of GIPC with endogenous NRP-1 and the cytoplasmic domain of NRP-1 (EGNP-1) in EC by coimmunoprecipitation. These data suggest that NRP-1 and GIPC may form an EC-special complex in vivo independent of VPF/VEGF. The interaction between EGNP-1 and GIPC advertises the existence of an independent signaling pathway for NRP-1-mediated VPF/VEGF function, which is against the current understanding that NRP-1 signaling is mainly conveyed by its extracellular domain. Indeed, in this study, we demonstrated that deletion of the C-terminal three amino acids of NRP-1 (SEA-COOH) abolished the interaction with GIPC in EC. Taken together, our data support our hypothesis that C-terminal three amino acids of NRP-1 are key functional domains, which may function by binding the PDZ domain of GIPC in a vascular EC line.

In zebrafish, the expression of zGIPC was detected in CNS and developing vessels, which is consistent with the expression pattern of zNRP-1 (6) . The reduction of zGIPC resulted in similar vessel defects to the reduction of zNRP-1, which suggests that zGIPC may function in the same signaling pathway as the NRP-1. This notion was further confirmed by an in vitro assay to measure the EC migration, a critical event in angiogenesis. Our previous data in HUVECs indicated that NRP-1 alone is required for EC migration (12) . We showed that blocking GIPC expression inhibited HUVECs migration mediated by NRP-1/EGNP-1. Interestingly, we found that overexpresssion of hGIPC in zebrafish results in vessel overgrowth, a phenomenon that may be of importance in hemangiomas and vascular malformation. In summary, we demonstrated that GIPC is a key adapter protein to convey NRP-1-mediated signaling to regulate angiogenesis processes such as EC migration.

This study revealed an important role for the PDZ domain of GIPC in angiogenesis. We showed that injection of mRNA encoding a mutated human GIPC with a deleted PDZ domain cannot rescue the knockdown phenotype of zGIPC. In fact, injection of mRNA encoding such a mutated hGIPC alone resulted in similar phenotypes to reduction of zGIPC, which suggested that the deletion of PDZ domain may prevent the recruitment of downstream signaling components into the NRP-1 receptor complex and thus block the angiogenesis in a dominant negative manner. These results provide genetic evidence to support the essential role of the interaction between the PDZ domain of GIPC and the C-terminal three amino acids of NRP-1 (SEA-COOH).

On the basis of our data, we can speculate the molecular mechanisms on how GIPC convey NRP-1 signaling in angiogenesis. GIPC has be shown to link to G-protein signaling pathways by directly binding with G{alpha} interacting protein (GAIP), a member of the regulator of G-protein signaling (RGS) protein family (21) . As suggested by its name, GAIP specifically interacts with the heterotrimeric G protein G{alpha}i3 (47 48 49 50 51 52 53) . Interestingly, its C terminus possesses a modified PDZ domain-binding motif (SEA) that can interact with the PDZ domain of GIPC (21) . Thus, it is logical to speculate that GIPC and GAIP provide a possible linkage between G protein signaling and NRP-1 in vascular ECs. We will test this hypothesis in future studies.

In summary, this study confirms that the C-terminal three amino acids of NRP-1 (SEA-COOH) are key functional domains for NRP-1-mediated angiogenesis. For the first time, we identified GIPC as an adaptor protein to interact with the C-terminal three amino acids of NRP-1 (SEA-COOH) through its PDZ domain and then convey the NRP-1-mediated EC signaling to regulate angiogenesis events such as migration. Our data suggest a cascade of events for NRP-1-mediated VPF/VEGF-induced angiogenesis: VPF/VEGF ->NRP -> GIPC -> G protein -> EC migration -> angiogenesis. By providing insights regarding the role of NRP-1 in vascular development and angiogenesis, this study holds significant clinical implications with regard to cancer, cardiovascular diseases, and neurological disorders.

ACKNOWLEDGMENTS

We sincerely thank to Drs Michael Klagsbrun and Marilyn G. Farquhar for support and Dr. Ascoli for GIPC cDNA and its PDZ mutant. This work is partly supported by National Institutes of Health Grants CA-78383, HL-072178, HL-70567, and a Grant from American Cancer Society to D. Mukhopadhyay, and by Fraternal Order of Eagles Cancer Research Fund #233 to X. Xu. L. Wang is a fellow of American Heart Association.

FOOTNOTES

2 These authors contributed equally to this work.

Received for publication December 2, 2005. Accepted for publication February 16, 2006.

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