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,1,2
* Department of Biochemistry and Molecular Biology,
Mayo Clinic Cancer Center, Mayo Clinic College of Medicine, Rochester, Minnesota, USA
1 Correspondence: D.M., Department Biochemistry and Molecular Biology, Gugg 1401A, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905, USA. 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
SPECIFIC AIMS
Neuropilin-1 (NRP-1) is one of the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) receptors that is involved in normal vascular development as well as pathological angiogenesis, but the mechanism is unclear. Initially, it was thought that there was an absence of intracellular signaling by VPF/VEGF through NRP-1 alone. 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. Therefore, the aim of this work is to elucidate the signal pathways mediated by NRP-1 and identify its downstream molecules and its functional role in angiogenesis.
PRINCIPLE FINDINGS
1. C-terminal three amino acids of NRP-1 (SEA-COOH) is 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 it might be a key functional domain for NRP-1 signaling. Since the in vivo process of angiogenesis in zebrafish has been well documented, we would like to investigate the functions of the C-terminal three amino acids (SEA-COOH) of NRP-1 in zebrafish. To confirm the functional importance of the C-terminal domain of NRP-1, as suggested by our in vitro studies, we injected hNRP-1
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-1SEA 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 dorsal longitudinal anastomotic vessel (DLAV), intersegmental vessel (ISV), and subintestinal vein (SIV; Fig. 1
), lack of blood flow in these vessels, and more significant reduction in body size. These data suggest that hNRP-1SEA may function as a dominant negative mutant. Indeed, injection of hNRP-1SEA alone disrupted the formation of DLAV, ISV, and SIV, which resembles the phenotypes generated from zNRP-1 knockdown (Fig. 1)
. As expected, injection of hNRP-1 mRNA showed normal vessel development. Hence, our 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.
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2. C-terminal domain of NRP-1 interacts with RGS-GAIP-interacting protein C terminus in endothelial cells
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 endothelial cells (ECs) through its C-terminal domain by interacting with C terminus of RGS-GAIP-interacting protein (GIPC). To test this hypothesis, we initially sought to confirm the protein-protein interaction between NRP-1 and GIPC in human umbilical vein endothelial cells (HUVECs). We characterized the expression of GIPC in HUVECs by using a polyclonal antibody against GIPC protein. By immunoprecipitation and immunoblotting, anti-GIPC antibody (Ab) detected a specific band with molecular masses 
40 kDa, which corresponds to the size of GIPC found in the nervous system (39 kDa). Meanwhile, a coimmunoprecipitation experiment was also performed to confirm the physical interaction between endogenous NRP-1 and GIPC. Moreover, when HUVEC cell lysates were immunoprecipitated with an anti-NRP-1 Ab followed by immunoblotting with anti-GIPC Ab, GIPC coimmunoprecipitated with NRP-1. Furthermore, we were able to show that the chimeric receptor of NRP-1, EGNP-1, could interact with GIPC, but deletion of the C-terminal three residues of NRP-1 (EGNP-1SEA) 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 of NRP-1 with GIPC is mediated by its C-terminal three amino acids (SEA-COOH) in vascular endothelium.
3. GIPC expresses in zebrafish vasculature and is required for angiogenesis by involving in NRP-1-mediating EC migration
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 (data not shown). The expression patterns of zGIPC during embryogenesis were characterized by whole mount in situ hybridization at 18-somite stage, 24 h postfertilization (hpf; zebrafish embryonic stages) and 48 hpf. In the embryos of 18-somite and 24 and 48 hpf, the expression of zGIPC was observed in the brain and probably the central nervous system, which is consistent with the previous finding in the mouse. Interestingly, the GIPC transcript was detected in embryo 18-somite stages in the trunk region; two bilateral stripes of GIPC-positive cells reached the tailed region and converged toward the ventral midline to merge into a single stripe and terminated at the ventral region of the tail (Fig. 2
A). 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. 2A
). The transient expression pattern is consistent with those from NRP-1a and 1b. To analyze the function of zGIPC during zebrafish angiogenesis, we knocked down zGIPC expression using morpholino antisense oligoes. Injection of 1.25 ng zGIPC morpholino into TG(fli1:egfp) embryos disturbed the formation of DLAV and SIV. Injection of 10 ng zGIPC morpholino results in more severe vessel defects including DLAV, ISV, and SIV (Fig. 2B
). 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 the NRP-1 signaling pathway. To address the functions of GIPC in NRP-1 signaling, we analyzed the role of GIPC in NRP-1/EGNP-1-mediated HUVEC migration. For this purpose, we first knocked down GIPC in the HUVECs by using RNA interference-mediated silencing and performed migration assays. Interestingly, after 2 h of incubation, EC migration induced by epidermal growth factor (EGF) in EGNP-1-transduced HUVEC increased 2-fold than that of untransfected HUVECs or HUVECs transduced with LacZ. 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 a Student’s t test, compare 0.03, 0.3, and 3 pM GIPC siRNA silencing groups with no silencing group, respectively, in EGNP-1 transduced HUVECs), whereas the control siRNA did not (P value is 0.0557 in a Student’s t test). HUVECs transduced by increasing amounts of GIPC siRNA progressively blocked migration of EC. Inconsistent with our in vivo data, these data further suggested that GIPC is involved in NRP-1-mediated EC migration.
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4. PDZ domain of GIPC is a key functional domain in GIPC-mediated NRP-1-induced angiogenesis
Furthermore, the role of the PDZ domain of GIPC in angiogenesis was explored in this study. Coinjection of hGIPC mRNA with zGIPC morpholino rescued the phenotype of zGIPC knockdown and thus displayed normal vessel and circulation. However, coinjection of hGIPCPDZ mRNA with zGIPC morpholino did not rescue zGIPC knockdown phenotype despite similar levels of protein expression. 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).
CONCLUSIONS AND SIGNIFICANCE
This study showed for the first time that the C-terminal three amino acids of NRP-1 (SEA-COOH) are key functional domains for NRP-1-mediated angiogenesis. Moreover, this study identifies that the C-terminal three amino acids of NRP-1 (SEA-COOH) can interact with GIPC in ECs and reveals that GIPC can mediate the signaling steps necessary by its PDZ domain for NRP-1-mediated angiogenesis, especially EC migration event. These findings support our current model for NRP-1-mediated VPF/VEGF-induced angiogenesis (Fig. 3
). Overall, this study provides more insights regarding the role of NRP-1 in vascular development and angiogenesis and, therefore, holds significant clinical applications for cancers, cardiovascular diseases, and neurological disorders.
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FOOTNOTES
2 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5504fje
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