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Neuropilin-1 functions as a VEGFR2 co-receptor to guide developmental angiogenesis independent of ligand binding.

Gelfand MV, Hagan N, Tata A, Oh WJ, Lacoste B, Kang KT, Kopycinska J, Bischoff J, Wang JH, Gu C - Elife (2014)

Bottom Line: In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1(VEGF-)).Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2.Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Harvard Medical School, Boston, United States.

ABSTRACT
During development, tissue repair, and tumor growth, most blood vessel networks are generated through angiogenesis. Vascular endothelial growth factor (VEGF) is a key regulator of this process and currently both VEGF and its receptors, VEGFR1, VEGFR2, and Neuropilin1 (NRP1), are targeted in therapeutic strategies for vascular disease and cancer. NRP1 is essential for vascular morphogenesis, but how NRP1 functions to guide vascular development has not been completely elucidated. In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1(VEGF-)). Nrp1(VEGF-) mutants survive to adulthood with normal vasculature revealing that NRP1 functions independent of VEGF-NRP1 binding during developmental angiogenesis. Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2. Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

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Assessment of additional Nrp1 variants containing mutations in the VEGF-binding site.AP-SEMA3A or AP-VEGF was applied to COS-1 cells overexpressing the indicated construct (top and middle row). Non-permeabilized antibody staining was performed with a polyclonal anti-NRP1 antibody to detect NRP1 surface expression (bottom row). Scale bar: 50 μm.DOI:http://dx.doi.org/10.7554/eLife.03720.004
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fig1s1: Assessment of additional Nrp1 variants containing mutations in the VEGF-binding site.AP-SEMA3A or AP-VEGF was applied to COS-1 cells overexpressing the indicated construct (top and middle row). Non-permeabilized antibody staining was performed with a polyclonal anti-NRP1 antibody to detect NRP1 surface expression (bottom row). Scale bar: 50 μm.DOI:http://dx.doi.org/10.7554/eLife.03720.004

Mentions: We sought to elucidate the in vivo function of VEGF-NRP1 binding by generating a mouse line that selectively disrupts VEGF binding to NRP1. A previous structure–function analysis revealed that the b1 domain of NRP1 is necessary and sufficient for VEGF binding (Gu et al., 2002). However, this b1 region is also required for SEMA3-NRP1 interactions, so a series of Nrp1 variants containing smaller deletions in the b1 domain were engineered with site-directed mutagenesis to identify a region specific for VEGF-NRP1 binding (Figure 1A). Based upon previous publications, we first targeted two specific sites in the b1 domain: the 7-residue binding site of the Pathologische Anatomie Leiden-Endothelium (PAL-E) monoclonal antibody which competes with VEGF for NRP1 binding (Jaalouk et al., 2007) and the 3-residue binding site of the VEGF analog tuftsin (Vander Kooi et al., 2007) (Figure 1A–B). COS-1 cells were transfected with wild-type (WT) or mutant Nrp1 constructs and assessed for NRP1 expression. PAL-E and tuftsin binding site mutations did not affect NRP1 protein expression at the cell surface as examined by non-permeabilized antibody staining (Figure 1C, Figure 1—figure supplement 1). Ligand binding to NRP1 was assessed using alkaline phosphatase-tagged VEGF (AP-VEGF) and SEMA3A (AP-SEMA3A) in conjunction with alkaline phosphatase histochemistry. All of the PAL-E or tuftsin binding site variants were capable of abolishing VEGF-NRP1 binding, but unfortunately, also eliminated SEMA3-NRP1 binding (Figure 1C, Figure 1—figure supplement 1).10.7554/eLife.03720.003Figure 1.Design and assessment of Nrp1 variants harboring mutations in the VEGF-binding site.


Neuropilin-1 functions as a VEGFR2 co-receptor to guide developmental angiogenesis independent of ligand binding.

Gelfand MV, Hagan N, Tata A, Oh WJ, Lacoste B, Kang KT, Kopycinska J, Bischoff J, Wang JH, Gu C - Elife (2014)

Assessment of additional Nrp1 variants containing mutations in the VEGF-binding site.AP-SEMA3A or AP-VEGF was applied to COS-1 cells overexpressing the indicated construct (top and middle row). Non-permeabilized antibody staining was performed with a polyclonal anti-NRP1 antibody to detect NRP1 surface expression (bottom row). Scale bar: 50 μm.DOI:http://dx.doi.org/10.7554/eLife.03720.004
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4197402&req=5

fig1s1: Assessment of additional Nrp1 variants containing mutations in the VEGF-binding site.AP-SEMA3A or AP-VEGF was applied to COS-1 cells overexpressing the indicated construct (top and middle row). Non-permeabilized antibody staining was performed with a polyclonal anti-NRP1 antibody to detect NRP1 surface expression (bottom row). Scale bar: 50 μm.DOI:http://dx.doi.org/10.7554/eLife.03720.004
Mentions: We sought to elucidate the in vivo function of VEGF-NRP1 binding by generating a mouse line that selectively disrupts VEGF binding to NRP1. A previous structure–function analysis revealed that the b1 domain of NRP1 is necessary and sufficient for VEGF binding (Gu et al., 2002). However, this b1 region is also required for SEMA3-NRP1 interactions, so a series of Nrp1 variants containing smaller deletions in the b1 domain were engineered with site-directed mutagenesis to identify a region specific for VEGF-NRP1 binding (Figure 1A). Based upon previous publications, we first targeted two specific sites in the b1 domain: the 7-residue binding site of the Pathologische Anatomie Leiden-Endothelium (PAL-E) monoclonal antibody which competes with VEGF for NRP1 binding (Jaalouk et al., 2007) and the 3-residue binding site of the VEGF analog tuftsin (Vander Kooi et al., 2007) (Figure 1A–B). COS-1 cells were transfected with wild-type (WT) or mutant Nrp1 constructs and assessed for NRP1 expression. PAL-E and tuftsin binding site mutations did not affect NRP1 protein expression at the cell surface as examined by non-permeabilized antibody staining (Figure 1C, Figure 1—figure supplement 1). Ligand binding to NRP1 was assessed using alkaline phosphatase-tagged VEGF (AP-VEGF) and SEMA3A (AP-SEMA3A) in conjunction with alkaline phosphatase histochemistry. All of the PAL-E or tuftsin binding site variants were capable of abolishing VEGF-NRP1 binding, but unfortunately, also eliminated SEMA3-NRP1 binding (Figure 1C, Figure 1—figure supplement 1).10.7554/eLife.03720.003Figure 1.Design and assessment of Nrp1 variants harboring mutations in the VEGF-binding site.

Bottom Line: In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1(VEGF-)).Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2.Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Harvard Medical School, Boston, United States.

ABSTRACT
During development, tissue repair, and tumor growth, most blood vessel networks are generated through angiogenesis. Vascular endothelial growth factor (VEGF) is a key regulator of this process and currently both VEGF and its receptors, VEGFR1, VEGFR2, and Neuropilin1 (NRP1), are targeted in therapeutic strategies for vascular disease and cancer. NRP1 is essential for vascular morphogenesis, but how NRP1 functions to guide vascular development has not been completely elucidated. In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1(VEGF-)). Nrp1(VEGF-) mutants survive to adulthood with normal vasculature revealing that NRP1 functions independent of VEGF-NRP1 binding during developmental angiogenesis. Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2. Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

Show MeSH
Related in: MedlinePlus