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Neuropilin-1/GIPC1 signaling regulates alpha5beta1 integrin traffic and function in endothelial cells.

Valdembri D, Caswell PT, Anderson KI, Schwarz JP, König I, Astanina E, Caccavari F, Norman JC, Humphries MJ, Bussolino F, Serini G - PLoS Biol. (2009)

Bottom Line: Furthermore, it has been recently reported that Nrp1 is required for endothelial cell (EC) response to both VEGF-A165 and VEGF-A121 isoforms, the latter being incapable of binding Nrp1 on the EC surface.Binding of the homomultimeric endocytic adaptor GAIP interacting protein C terminus, member 1 (GIPC1), to the SEA motif of Nrp1 selectively stimulates the internalization of active alpha5beta1 in Rab5-positive early endosomes.In conclusion, we propose that Nrp1, in addition to and independently of its role as coreceptor for VEGF-A165 and SEMA3A, stimulates through its cytoplasmic domain the spreading of ECs on fibronectin by increasing the Rab5/GIPC1/Myo6-dependent internalization of active alpha5beta1.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncological Sciences and Division of Molecular Angiogenesis, Institute for Cancer Research and Treatment, University of Torino School of Medicine, Candiolo, Italy.

ABSTRACT
Neuropilin 1 (Nrp1) is a coreceptor for vascular endothelial growth factor A165 (VEGF-A165, VEGF-A164 in mice) and semaphorin 3A (SEMA3A). Nevertheless, Nrp1 embryos display vascular defects that differ from those of mice lacking either VEGF-A164 or Sema3A proteins. Furthermore, it has been recently reported that Nrp1 is required for endothelial cell (EC) response to both VEGF-A165 and VEGF-A121 isoforms, the latter being incapable of binding Nrp1 on the EC surface. Taken together, these data suggest that the vascular phenotype caused by the loss of Nrp1 could be due to a VEGF-A164/SEMA3A-independent function of Nrp1 in ECs, such as adhesion to the extracellular matrix. By using RNA interference and rescue with wild-type and mutant constructs, we show here that Nrp1 through its cytoplasmic SEA motif and independently of VEGF-A165 and SEMA3A specifically promotes alpha5beta1-integrin-mediated EC adhesion to fibronectin that is crucial for vascular development. We provide evidence that Nrp1, while not directly mediating cell spreading on fibronectin, interacts with alpha5beta1 at adhesion sites. Binding of the homomultimeric endocytic adaptor GAIP interacting protein C terminus, member 1 (GIPC1), to the SEA motif of Nrp1 selectively stimulates the internalization of active alpha5beta1 in Rab5-positive early endosomes. Accordingly, GIPC1, which also interacts with alpha5beta1, and the associated motor myosin VI (Myo6) support active alpha5beta1 endocytosis and EC adhesion to fibronectin. In conclusion, we propose that Nrp1, in addition to and independently of its role as coreceptor for VEGF-A165 and SEMA3A, stimulates through its cytoplasmic domain the spreading of ECs on fibronectin by increasing the Rab5/GIPC1/Myo6-dependent internalization of active alpha5beta1. Nrp1 modulation of alpha5beta1 integrin function can play a causal role in the generation of angiogenesis defects observed in Nrp1 mice.

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Nrp1 Regulates the Traffic of Active α5β1 Integrin in ECs(A) Time-course assays reveal an impairment of active but not total α5β1 integrin internalization in ECs silenced for hNrp1 (sihN1) in comparison with cells transfected with control siRNA (siCtl).(B,C) Relative quantifications of time-course internalization assays shown in (A) of total (B) and active (C) α5β1 integrin are depicted.(D) Wild-type mNrp1, but neither mNrp1dSEA nor mNrp1dCy deletion constructs, was able to rescue the early (4 min) internalization defects of active α5β1 integrin in sihNrp1 ECs as quantified in the lower histogram.
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pbio-1000025-g006: Nrp1 Regulates the Traffic of Active α5β1 Integrin in ECs(A) Time-course assays reveal an impairment of active but not total α5β1 integrin internalization in ECs silenced for hNrp1 (sihN1) in comparison with cells transfected with control siRNA (siCtl).(B,C) Relative quantifications of time-course internalization assays shown in (A) of total (B) and active (C) α5β1 integrin are depicted.(D) Wild-type mNrp1, but neither mNrp1dSEA nor mNrp1dCy deletion constructs, was able to rescue the early (4 min) internalization defects of active α5β1 integrin in sihNrp1 ECs as quantified in the lower histogram.

Mentions: The efficiency of cell adhesion and spreading on ECM is generally thought to be proportional to the amount of either active or total (i.e., active and inactive) integrin at the cell surface [1,6]. We found that lack of Nrp1 did not alter the global amount of either total (Figure 1A), as already reported [31], or active α5β1 integrin, as recognized by the mouse monoclonal Ab (mAb) SNAKA51 [45] (Figure S3). Then, we analyzed whether Nrp1 could influence the amount of α5β1 integrin on the endothelial surface. Biotinylation experiments revealed that knocking down human Nrp1 did not diminish the surface levels of either total or active α5β1 integrin in sihNrp1 ECs (Figure 6A), thus suggesting that a mechanism alternative to the control of integrin conformation should be responsible for Nrp1-dependent activation of α5β1 integrin function in ECs.


Neuropilin-1/GIPC1 signaling regulates alpha5beta1 integrin traffic and function in endothelial cells.

Valdembri D, Caswell PT, Anderson KI, Schwarz JP, König I, Astanina E, Caccavari F, Norman JC, Humphries MJ, Bussolino F, Serini G - PLoS Biol. (2009)

Nrp1 Regulates the Traffic of Active α5β1 Integrin in ECs(A) Time-course assays reveal an impairment of active but not total α5β1 integrin internalization in ECs silenced for hNrp1 (sihN1) in comparison with cells transfected with control siRNA (siCtl).(B,C) Relative quantifications of time-course internalization assays shown in (A) of total (B) and active (C) α5β1 integrin are depicted.(D) Wild-type mNrp1, but neither mNrp1dSEA nor mNrp1dCy deletion constructs, was able to rescue the early (4 min) internalization defects of active α5β1 integrin in sihNrp1 ECs as quantified in the lower histogram.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2631072&req=5

pbio-1000025-g006: Nrp1 Regulates the Traffic of Active α5β1 Integrin in ECs(A) Time-course assays reveal an impairment of active but not total α5β1 integrin internalization in ECs silenced for hNrp1 (sihN1) in comparison with cells transfected with control siRNA (siCtl).(B,C) Relative quantifications of time-course internalization assays shown in (A) of total (B) and active (C) α5β1 integrin are depicted.(D) Wild-type mNrp1, but neither mNrp1dSEA nor mNrp1dCy deletion constructs, was able to rescue the early (4 min) internalization defects of active α5β1 integrin in sihNrp1 ECs as quantified in the lower histogram.
Mentions: The efficiency of cell adhesion and spreading on ECM is generally thought to be proportional to the amount of either active or total (i.e., active and inactive) integrin at the cell surface [1,6]. We found that lack of Nrp1 did not alter the global amount of either total (Figure 1A), as already reported [31], or active α5β1 integrin, as recognized by the mouse monoclonal Ab (mAb) SNAKA51 [45] (Figure S3). Then, we analyzed whether Nrp1 could influence the amount of α5β1 integrin on the endothelial surface. Biotinylation experiments revealed that knocking down human Nrp1 did not diminish the surface levels of either total or active α5β1 integrin in sihNrp1 ECs (Figure 6A), thus suggesting that a mechanism alternative to the control of integrin conformation should be responsible for Nrp1-dependent activation of α5β1 integrin function in ECs.

Bottom Line: Furthermore, it has been recently reported that Nrp1 is required for endothelial cell (EC) response to both VEGF-A165 and VEGF-A121 isoforms, the latter being incapable of binding Nrp1 on the EC surface.Binding of the homomultimeric endocytic adaptor GAIP interacting protein C terminus, member 1 (GIPC1), to the SEA motif of Nrp1 selectively stimulates the internalization of active alpha5beta1 in Rab5-positive early endosomes.In conclusion, we propose that Nrp1, in addition to and independently of its role as coreceptor for VEGF-A165 and SEMA3A, stimulates through its cytoplasmic domain the spreading of ECs on fibronectin by increasing the Rab5/GIPC1/Myo6-dependent internalization of active alpha5beta1.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncological Sciences and Division of Molecular Angiogenesis, Institute for Cancer Research and Treatment, University of Torino School of Medicine, Candiolo, Italy.

ABSTRACT
Neuropilin 1 (Nrp1) is a coreceptor for vascular endothelial growth factor A165 (VEGF-A165, VEGF-A164 in mice) and semaphorin 3A (SEMA3A). Nevertheless, Nrp1 embryos display vascular defects that differ from those of mice lacking either VEGF-A164 or Sema3A proteins. Furthermore, it has been recently reported that Nrp1 is required for endothelial cell (EC) response to both VEGF-A165 and VEGF-A121 isoforms, the latter being incapable of binding Nrp1 on the EC surface. Taken together, these data suggest that the vascular phenotype caused by the loss of Nrp1 could be due to a VEGF-A164/SEMA3A-independent function of Nrp1 in ECs, such as adhesion to the extracellular matrix. By using RNA interference and rescue with wild-type and mutant constructs, we show here that Nrp1 through its cytoplasmic SEA motif and independently of VEGF-A165 and SEMA3A specifically promotes alpha5beta1-integrin-mediated EC adhesion to fibronectin that is crucial for vascular development. We provide evidence that Nrp1, while not directly mediating cell spreading on fibronectin, interacts with alpha5beta1 at adhesion sites. Binding of the homomultimeric endocytic adaptor GAIP interacting protein C terminus, member 1 (GIPC1), to the SEA motif of Nrp1 selectively stimulates the internalization of active alpha5beta1 in Rab5-positive early endosomes. Accordingly, GIPC1, which also interacts with alpha5beta1, and the associated motor myosin VI (Myo6) support active alpha5beta1 endocytosis and EC adhesion to fibronectin. In conclusion, we propose that Nrp1, in addition to and independently of its role as coreceptor for VEGF-A165 and SEMA3A, stimulates through its cytoplasmic domain the spreading of ECs on fibronectin by increasing the Rab5/GIPC1/Myo6-dependent internalization of active alpha5beta1. Nrp1 modulation of alpha5beta1 integrin function can play a causal role in the generation of angiogenesis defects observed in Nrp1 mice.

Show MeSH
Related in: MedlinePlus