Cloning and characterization of neuropilin-1-interacting protein: a PSD-95/Dlg/ZO-1 domain-containing protein that interacts with the cytoplasmic domain of neuropilin-1

Abstract

Neuropilin-1 (Npn-1), a receptor for semaphorin III, mediates the guidance of growth cones on extending neurites. The molecular mechanism of Npn-1 signaling remains unclear. We have used a yeast two-hybrid system to isolate a protein that interacts with the cytoplasmic domain of Npn-1. This Npn-1-interacting protein (NIP) contains a central PSD-95/Dlg/ZO-1 (PDZ) domain and a C-terminal acyl carrier protein domain. The physiological interaction of Npn-1 and NIP is supported by co-immunoprecipitation of these two proteins in extracts from a heterologous expression system and from a native tissue. The C-terminal three amino acids of Npn-1 (S-E-A-COOH), which is conserved from Xenopus to human, is responsible for interaction with the PDZ domain-containing C-terminal two-thirds of NIP. NIP as well as Npn-1 are broadly expressed in mice as assayed by Northern and Western analysis. Immunohistochemistry and in situ hybridization experiments revealed that NIP expression overlaps with that of Npn-1. NIP has been independently cloned as RGS-GAIP-interacting protein (GIPC), where it was identified by virtue of its interaction with the C terminus of RGS-GAIP and suggested to participate in clathrin-coated vesicular trafficking. We suggest that NIP and GIPC may participate in regulation of Npn-1-mediated signaling as a molecular adapter that couples Npn-1 to membrane trafficking machinery in the dynamic axon growth cone.

Fig. 1.

Domain structure of Npn-1 and sequence alignment of Npn-1 cytoplasmic domains. Npn-1s share ∼70 and 90% amino acid identities in the extracellular and cytoplasmic domains, respectively. The amino acid sequences of cytoplasmic domains of Npn-1 (Xenopus, chick, mouse, rat, and human), and Npn-2a (rat) are aligned, and the identical amino acids are shown in bold.TM, Transmembrane domain, Cy, cytoplasmic domain.

Fig. 2.

Co-immunoprecipitation of NIP and Npn-1.A, The full-length N-terminal Myc-tagged Npn-1 with or without HA-tagged NIP were expressed in HEK 293T cells and immunoprecipitated with an anti-Myc antibody. The anti-NIP antibody was used to detect the NIP protein. In the bottom two panels, aliquots of whole-cell extract were immunoblotted with anti-Myc or anti-HA antibodies. Molecular size markers are indicated in kilodaltons. B, HA-tagged NIP was co-expressed with Myc-tagged full-length Npn-1 or mutated Npn-1 lacking the C-terminal three residues in HEK 293T cells. Extracts were subjected to immunoprecipitation with the anti-NIP peptide antibody or preimmune serum. The resulting immunoprecipitates were probed with anti-Myc antibody to detect Npn-1 and its mutant form. In the bottom two panels, aliquots of total cell extracts were immunoblotted with anti-Myc or anti-HA antibod ies to confirm the similar expression levels of the constructs. C, NIP was immunoprecipitated from solubilized olfactory bulb membrane extract (input) by using the NIP peptide antibody and the resulting immunoprecipitates were probed with a Npn-1 antibody. Npn-1 was co-immunoprecipitated with NIP. Preimmune serum failed to immunoprecipitated Npn-1.

Fig. 3.

Distribution of NIP protein in postnatal day 2 mouse tissues. A, The NIP anti-fusion protein and the anti-peptide antibodies recognize a 40 kDa protein in the olfactory bulb whole-cell lysate. Detection of the NIP protein by the anti-peptide antibody was blocked by preincubation with specific peptide. Preimmune serum failed to detect the NIP protein.B, Immunoblot of 11 tissues revealed a broad tissue distribution of NIP protein. Equal amounts of whole tissue lysates (50 μg of proteins) were separated by gel electorphoresis and probed with the anti-peptide antibody.

Fig. 4.

Expression of NIP mRNA in neurons of the embryonic mouse spinal cord and DRG. NIP mRNA is expressed in neurons of the dorsal and ventral spinal cord as well as DRG revealed by in situ hybridization on a transverse spinal cord section of an E14 embryo using digoxigenin-labeled NIP riboprobe (A). The sense probe failed to detect any signals in the adjacent section (B). DH, Dorsal horn of spinal cord; VH, ventral horn of spinal cord.

Fig. 5.

Expression of Npn-1 and NIP protein in the embryonic mouse CNS and PNS. Both Npn-1 and NIP proteins are expressed in E14 superior cervical ganglion neurons (SCG) (A, B), the optic nerve (ON) (D, E), the olfactory axon (AX) underneath the olfactory epithelium (OE), and the olfactory nerve layer (ONL) terminating at the surface of olfactory bulb (OB) (G, H). The sections were stained with anti-Npn-1 (A, D, G) and anti-NIP (B, E, H) antibodies. Preimmune serum failed to detect signals (C, F, I).

Fig. 6.

Heterogeneous expression of NIP and Npn-1 proteins in the adult rat olfactory nerve bundles and terminals. Transverse sections of adult rat olfactory epithelium and bulb were immunostained with the anti-NIP and anti-Npn-1 antibodies. Npn-1 staining was detected predominantly in the olfactory axon bundles (AX) underneath the olfactory epithelium (A) and also in the olfactory neuron layers (ORL) and glomeruli (GLO) (C), but not in the sustentacular cell layer (SCL) and basal cell layer (BCL). NIP staining was detected exclusively in the olfactory axon bundles underneath the olfactory epithelium (B) and at the surface of the olfactory bulb covered by olfactory nerve layer and glomeruli (D). Both Npn-1 and NIP immunoreactivities were detected in olfactory nerve terminals within the same subset of glomeruli on two adjacent olfactory bulb sections (C, D). Arrowheads point to the glomeruli that show weaker staining with both anti-Npn-1 and anti-NIP antibodies (C, D).