Characterization of Neuropilin-1 Structural Features That Confer Binding to Semaphorin 3A and Vascular Endothelial Growth Factor 165*

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.

[1]  M. Tessier-Lavigne,et al.  Neuropilin Is a Receptor for the Axonal Chemorepellent Semaphorin III , 1997, Cell.

[2]  D. Wilkinson,et al.  Eph receptors and ephrins: regulators of guidance and assembly. , 2000, International review of cytology.

[3]  David J. Anderson,et al.  Molecular Distinction and Angiogenic Interaction between Embryonic Arteries and Veins Revealed by ephrin-B2 and Its Receptor Eph-B4 , 1998, Cell.

[4]  D. Anderson,et al.  Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development. , 1999, Molecular cell.

[5]  C. Mailhos,et al.  Vascular developmental biology: getting nervous. , 2000, Current opinion in genetics & development.

[6]  H. Fujisawa,et al.  Determination of Cell Adhesion Sites of Neuropilin-1 , 2000, The Journal of cell biology.

[7]  R. Klein,et al.  Eph receptors and ephrins: effectors of morphogenesis. , 1999, Development.

[8]  T. Yagi,et al.  Neuropilin–Semaphorin III/D-Mediated Chemorepulsive Signals Play a Crucial Role in Peripheral Nerve Projection in Mice , 1997, Neuron.

[9]  Alex L Kolodkin,et al.  Neuropilin Is a Semaphorin III Receptor , 1997, Cell.

[10]  Alex L Kolodkin,et al.  Neuropilin-2 Is a Receptor for Semaphorin IV Insight into the Structural Basis of Receptor Function and Specificity , 1998, Neuron.

[11]  Zhigang He,et al.  Semaphorin–Neuropilin Interactions Underlying Sympathetic Axon Responses to Class III Semaphorins , 1998, Neuron.

[12]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[13]  S. Takagi,et al.  Expression of a cell adhesion molecule, neuropilin, in the developing chick nervous system. , 1995, Developmental biology.

[14]  J. Bolz,et al.  Semaphorin 3A–Vascular Endothelial Growth Factor-165 Balance Mediates Migration and Apoptosis of Neural Progenitor Cells by the Recruitment of Shared Receptor , 2001, The Journal of Neuroscience.

[15]  M. Tessier-Lavigne,et al.  Plexin-A3 Mediates Semaphorin Signaling and Regulates the Development of Hippocampal Axonal Projections , 2001, Neuron.

[16]  N. Ferrara.  Role of vascular endothelial growth factor in regulation of physiological angiogenesis. , 2001, American journal of physiology. Cell physiology.

[17]  T. Yagi,et al.  A requirement for neuropilin-1 in embryonic vessel formation. , 1999, Development.

[18]  J. Epstein,et al.  Targeted disruption of semaphorin 3C leads to persistent truncus arteriosus and aortic arch interruption. , 2001, Development.

[19]  M. Poo,et al.  Unified Nomenclature for the Semaphorins/Collapsins , 1999, Cell.

[20]  H J Motulsky,et al.  Fitting curves to data using nonlinear regression: a practical and nonmathematical review , 1987, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[21]  Axel T. Brunger,et al.  X-PLOR Version 3.1: A System for X-ray Crystallography and NMR , 1992 .

[22]  G. Neufeld,et al.  Vascular endothelial growth factor (VEGF) and its receptors , 1999, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[23]  R. Kalb,et al.  Plexin-Neuropilin-1 Complexes Form Functional Semaphorin-3A Receptors , 1999, Cell.

[24]  T. Amagai,et al.  Specific cell surface labels in the visual centers of Xenopus laevis tadpole identified using monoclonal antibodies. , 1987, Developmental biology.

[25]  L. Feiner,et al.  A Dominant Negative Receptor for Specific Secreted Semaphorins Is Generated by Deleting an Extracellular Domain from Neuropilin-1 , 1999, The Journal of Neuroscience.

[26]  J. Calvete,et al.  The crystal structures of two spermadhesins reveal the CUB domain fold , 1997, Nature Structural Biology.

[27]  S V Evans,et al.  SETOR: hardware-lighted three-dimensional solid model representations of macromolecules. , 1993, Journal of molecular graphics.

[28]  R. Kalb,et al.  Molecular basis of semaphorin-mediated axon guidance. , 2000, Journal of neurobiology.

[29]  M. Kanje,et al.  Vascular endothelial growth factor is a neurotrophic factor which stimulates axonal outgrowth through the flk‐1 receptor , 2000, The European journal of neuroscience.

[30]  T. Kitsukawa,et al.  Developmentally regulated expression of a cell surface protein, neuropilin, in the mouse nervous system. , 1996, Journal of neurobiology.

[31]  P. Leder,et al.  The kit ligand: A cell surface molecule altered in steel mutant fibroblasts , 1990, Cell.

[32]  R. Kalb,et al.  Neuropilin-1 Extracellular Domains Mediate Semaphorin D/III-Induced Growth Cone Collapse , 1998, Neuron.

[33]  Shay Soker,et al.  Neuropilin-1 Is Expressed by Endothelial and Tumor Cells as an Isoform-Specific Receptor for Vascular Endothelial Growth Factor , 1998, Cell.

[34]  S. Strittmatter,et al.  PlexinA1 Autoinhibition by the Plexin Sema Domain , 2001, Neuron.

[35]  C. Goodman,et al.  The Molecular Biology of Axon Guidance , 1996, Science.

[36]  B. J. Limberg,et al.  Vascular Endothelial Growth Factor Receptor-2 and Neuropilin-1 Form a Receptor Complex That Is Responsible for the Differential Signaling Potency of VEGF165 and VEGF121 * , 2001, The Journal of Biological Chemistry.

[37]  T. Kitsukawa,et al.  Roles of a neuronal cell-surface molecule, neuropilin, in nerve fiber fasciculation and guidance , 1997, Cell and Tissue Research.

[38]  Jonathan A Raper,et al.  Semaphorins and their receptors in vertebrates and invertebrates , 2000, Current Opinion in Neurobiology.

[39]  M. Poo,et al.  Plexins Are a Large Family of Receptors for Transmembrane, Secreted, and GPI-Anchored Semaphorins in Vertebrates , 1999, Cell.

[40]  K. Agata,et al.  The A5 antigen, a candidate for the neuronal recognition molecule, has homologies to complement components and coagulation factors , 1991, Neuron.

[41]  S. Soker,et al.  Neuropilin-1 Mediates Collapsin-1/Semaphorin III Inhibition of Endothelial Cell Motility , 1999, The Journal of cell biology.