Identification of two novel alternatively spliced Neuropilin-1 isoforms

Abstract

Neuropilin-1 (NRP1) is a coreceptor to a tyrosine kinase receptor for both the vascular endothelial growth factor (VEGF) family and semaphorin (Sema) family members. NRP1 plays versatile roles in angiogenesis, axon guidance, cell survival, migration, and invasion. NRP1 contains three distinct extracellular domains, a1a2, b1b2, and c. We report here the identification of two novel soluble human NRP1 isoforms, which we named sIIINRP1 and sIVNRP1. These soluble NRP1 isoforms were generated by alternative splicing of the NRP1 gene, a common regulatory mechanism occurring in cell surface receptor families. Both sIIINRP1 and sIVNRP1 contain a1a2 and b1b2 domains, but no c domain, and the rest of the NRP1 sequence. Additionally, sIIINRP1 is missing 48 amino acids within the C-terminus of the b2 domain. Both sIIINRP1 and sIVNRP1 are expressed in human cancerous and normal tissues. These molecules are capable of binding to VEGF165 and Sema3A. Furthermore, recombinant sIIINRP1 and sIVNRP1 proteins inhibit NRP1-mediated MDA-MB-231 breast cancer cell migration. These results indicate the multiple levels of regulation in NRP1 function and suggest that these two novel NRP1 isoforms are useful antagonists for NRP1-mediated cellular activities.

Fig. 1

Structure of the full-length NRP1, the previously discovered soluble isoforms, and the novel soluble isoforms. (A) Genomic structure of the NRP1 gene as well as cDNA and protein structure of the full-length NRP1 and two previously identified soluble isoforms, s₁₂NRP1 and s₁₁NRP1 [19,20]. In the genomic structure, filled boxes represent translated exons, open boxes represent untranslated exons, lines represent introns. In the cDNA structures, each exon is represented by an open box. Intron-derived sequence is represented by a filled box. In the protein structures, each subdomain is represented by an oval or a rectangular box. Intron, exon, and subdomain sizes are not to scale. (B) cDNA and protein structures of novel soluble isoforms s_IIINRP1 and s_IVNRP1. Sequences at splice sites are shown, with exon-derived sequences in capitals and intron-derived sequences in lowercase. Splice donor and acceptor sites and each stop codon are underlined. The cleavage/polyadenylation site (aataaa) is in bold.

Fig. 2

Expression of s_IIINRP1 and s_IVNRP1 mRNA in various human normal and cancerous tissues and glioma cell lines. Expression of the NRP1 isoforms was examined in human tissues by RT-PCR using either primers capable of amplifying s₁₂NRP1, s_IIINRP1, and s_IVNRP1 simultaneously (A, top) or primers specific for each isoform (A, bottom, B, and C). Total RNA was isolated from various frozen human cancerous tissues and normal brains, or total RNA from various normal human tissues was purchased from Clontech. (A, top) Expression of three NRP1 isoforms (s₁₂NRP1, s_IIINRP1, and s_IVNRP1) in human glioma cell lines and normal brain. β-Actin primers as well as primers capable of amplifying s₁₂NRP1, s_IIINRP1, and s_IVNRP1 simultaneously were used. Sizes of RTPCR products are β-actin, 214 bp; s₁₂NRP1, 433 bp; s_IVNRP1, 328 bp; s_IIINRP1, 183 bp. (Bottom) Expression of s_IIINRP1 in identical glioma cell lines and normal brain tissues examined using a primer pair specific for s_IIINRP1. RT-PCR product of s_IIINRP1 is 114 bp. (B) Expression of s_IIINRP1 in various human primary glioma specimens, examined using a primer pair specific for s_IIINRP1. RT-PCR product of s_IIINRP1 is 114 bp. Glioma tissues are A, astrocytoma (WHO grade II); ODG, oligodendroglioma (WHO grade II); A + ODG, tumors showing morphology of both astrocytoma and oligodendroglioma (WHO grade II/III); PX, pleomorphic xanthoastrocytoma (WHO grade II/III); AA, anaplastic astrocytoma (WHO grade III); GBM, glioblastoma multiforme (WHO grade IV). (C) Expression of s_IVNRP1 in various human normal and cancerous tissues using a primer pair specific for s_IVNRP1. The RT-PCR product of s_IVNRP1 is 114 bp. Tissues used for analyses are lung carcinoid, lung adenocarcinoma (AdenoCA), lung squamous carcinoma (Squamous CA), AA, GBM, and normal kidney, lung, pancreas, and placenta tissues. All experiments shown in (A) to (C) were done at least two independent times with similar results.

Fig. 3

Expression of soluble NRP1 isoforms in COS-7 cells. Each soluble NRP1 isoform was assembled in a pcDNA 3.0 vector. The constructs were transfected separately into COS-7 cells with LipofectAMINE (Invitrogen). The lysates of various transfected cells were transferred to a membrane and immunoblotted with an anti-human NRP1 antibody (N-18; Santa Cruz Biotechnology). Protein of the correct size for each isoform was observed (s_IIINRP1, 60 kDa; s_IVNRP1, 67 kDa; and s₁₂NRP1, 90 kDa).

Fig. 4

Binding assays of soluble NRP1 isoforms and VEGF₁₆₅. (A) Qualitative assay of s_IIINRP1/VEGF₁₆₅ binding. CM containing s_IIINRP1, s_IVNRP1, s₁₂NRP1, VEGF₁₆₅-AP, proteins and Ig-basic-AP were produced by calcium phosphate transfection with various expression vectors into HEK 293T cells. CM containing serum was collected 48 h after transfection. CM containing soluble NRP1 was mixed with either CM containing VEGF₁₆₅-AP or CM from non-VEGF₁₆₅-AP-transfected cells (mock) and the assays for binding capacities of the soluble NRP1 isoforms were performed as described under Materials and methods. The sizes of the NRP1 proteins are as follows: s_IIINRP1, 60 kDa; s_IVNRP1, 67 kDa; s₁₂NRP1, 90 kDa. (B) Semiquantitative s_IIINRP1/VEGF₁₆₅-AP binding assay. Serum-free CM containing s_IIINRP1, s₁₂NRP1, and VEGF₁₆₅-AP proteins were produced in HEK 293T cells and quantification of the concentration of NRP1 or VEGF₁₆₅-AP proteins was done as described under Materials and methods. 3.0 nM VEGF₁₆₅-AP was with mixed with various concentrations (12.0, 6.0, 2.4, and 1.2 nM) of s₁₂NRP1 or s_IIINRP1 proteins along with protease inhibitors. The proteins were incubated at 4°C. An anti-AP antibody conjugated to agarose beads was added followed by further incubation. The precipitated proteins were transferred to a membrane and immunoblotted with an anti-NRP1 antibody. The sizes of soluble NRP1 proteins are s_IIINRP1, 60 kDa; and s₁₂NRP1, 90 kDa.

Fig. 5

Soluble forms of NRP1 inhibit VEGF₁₆₅-AP binding to MDA-MB-231 breast carcinoma cells. (A) VEGF₁₆₅-AP binding to MDA-MB-231 cells. Increasing amounts of VEGF₁₆₅-AP fusion proteins were preincubated in the presence of 1.0 μg/ml heparin for 30 min on ice followed by 2 h incubation with the MDA-MB-231 cells at 4°C. Cell-associated VEGF₁₆₅-AP was quantified by measuring the AP activity (absorbance at 420 nm) in the cell lysates. The VEGF₁₆₅-AP molar concentration was calculated from the AP activity as described under Materials and methods. (B) Soluble forms of NRP1 inhibit VEGF₁₆₅-AP binding to MDA-MB-231 cells. Equal concentrations (0.96, 4.8, or 24.0 nM) of s₁₂NRP1, s_IIINRP1, s_IVNRP1, or Ig-Basic-AP proteins were mixed with 0.48 nM VEGF₁₆₅-AP protein in the presence of 1.0 μg/ml heparin for 30 min on ice prior to cell binding. The change in bound VEGF₁₆₅-AP was calculated as the percentage of untreated control that has no soluble forms of NRP1 protein. The data shown are the mean values, and error bars indicate the SD. Experiments were performed in triplicate at two separate times and similar results were obtained.

Fig. 6

Soluble forms of NRP1 inhibit NRP1-mediated MDA-MB-231 breast carcinoma cell migration. MDA-MB-231 cells were preincubated with isotype-matched mouse IgG (25.0 mg/ml), anti-NRP1 antibody (A-12, 25.0 mg/ml), or equal concentrations of s₁₂NRP1, s_IIINRP1, s_IVNRP1, or Ig-Basic-AP (2.4 or 24.0 nM) in the presence of heparin (1.0 μg/ml) and ZVAD-FMK (an apoptosis inhibitor, 20.0 μg/ml) for 30 min on ice. Cells were allowed to migrate toward serum-free CM of NIH 3T3 cells. Inhibitory effects of soluble forms of NRP1 were indicated as percentage decrease in the number of migrating cells compared to that of the control cells in the absence of soluble forms of NRP1 protein. The data shown are the mean values, and error bars indicate the SD. Data were statistically significant (as determined by power analyses using one-way ANOVA, p < 0.001). Experiments were performed in triplicate two independent times and similar results were obtained.