Molecular dissection of NRG1-ERBB4 signaling implicates PTPRZ1 as a potential schizophrenia susceptibility gene

Abstract

Neuregulin and the neuregulin receptor ERBB4 have been genetically and functionally implicated in schizophrenia. In this study, we used the yeast two-hybrid system to identify proteins that interact with ERBB4, to identify genes and pathways that might contribute to schizophrenia susceptibility. We identified the MAGI scaffolding proteins as ERBB4-binding proteins. After validating the interaction of MAGI proteins with ERBB4 in mammalian cells, we demonstrated that ERBB4 expression, alone or in combination with ERBB2 or ERBB3, led to the tyrosine phosphorylation of MAGI proteins, and that this could be further enhanced with receptor activation by neuregulin. As MAGI proteins were previously shown to interact with receptor phosphotyrosine phosphatase beta/zeta (RPTPbeta), we postulated that simultaneous binding of MAGI proteins to RPTPbeta and ERBB4 forms a phosphotyrosine kinase/phosphotyrosine phosphatase complex. Studies in cultured cells confirmed both a spatial and functional association between ERBB4, MAGI and RPTPbeta. Given the evidence for this functional association, we examined the genes coding for MAGI and RPTPbeta for genetic association with schizophrenia in a Caucasian United Kingdom case-control cohort (n= approximately 1400). PTPRZ1, which codes for RPTPbeta, showed significant, gene-wide and hypothesis-wide association with schizophrenia in our study (best individual single-nucleotide polymorphism allelic P=0.0003; gene-wide P=0.0064; hypothesis-wide P=0.026). The data provide evidence for a role of PTPRZ1, and for RPTPbeta signaling abnormalities, in the etiology of schizophrenia. Furthermore, the data indicate a role for RPTPbeta in the modulation of ERBB4 signaling that may in turn provide further support for an important role of neuregulin/ERBB4 signaling in the molecular basis of schizophrenia.

Figure 1

Scheme of ERBB4-MAGI-RPTPβ interactions. PDZ domains of MAGI are numbered 0–5. ERBB4 binds to PDZ1, whereas RPTPβ binds to PDZ4. The line indicates the MAGI-2 sequence recovered in the yeast two-hybrid screen.

Figure 2

Co-precipitation of MAGI-2 and ERBB4. Cells were transfected with: (1) MAGI-2; (2) EICD (top panels) or ERBB4 (bottom panels); (3) vector; or (4) MAGI-2 and either EICD (top panels) or ERBB4 (bottom panels). Cells were lysed and subjected to immunoprecipitation (IP) with anti-HA antibody (to precipitate HA-tagged MAGI-2), and the amounts of EICD, ERBB4, or cleaved, C-terminal fragment of ERBB4 (CTF), were determined by immunoblotting (IB) with an antibody (C-18) against the C terminal of ERBB4 (left panels). In parallel, an aliquot of cell lysate from transfected cells (10% of the amounts used for the immunopreciptation) was immunoblotted to confirm expression of transfected proteins (right panels).

Figure 3

Co-localization of MAGI with ERBB4 and RPTPβ. (a) Colocalization of MAGI-2 with ERBB4. H4 cells were transfected with ERBB4, ERBB4 and HA-tagged MAGI-2, or ERBB4 EICD and HA-tagged MAGI-2 and the proteins visualized by immunocytochemistry with an antibody directed against the C-terminus of ERBB4 and with an anti-HA antibody. (b) Colocalization of endogenous MAGI-1 with ERBB4 and RPTPβ. ERBB4, MAGI and RPTPβ were visualized in rat oligodendrocyte progenitor cells (top) and premyelinating oligodendrocytes (bottom) using anti-ERBB4, anti-MAGI-1 and/or anti-RPTPβ antibodies.

Figure 4

Tyrosine-phosphorylation of MAGI proteins. (a) Phosphorylation of MAGI-1 by ERB-receptor kinases. Cells were transfected with MAGI-1, in the presence or absence of ERBB2, ERBB3 and ERBB4 and tyrosine phosphorylation of MAGI-1 determined. (b) Phosphorylation/dephosphorylation of MAGI-1. Cells were transfected with MAGI-1, ERBB2 and ERBB4, in the presence or absence of RPTPβ. Transfected cells were treated with vehicle or NRG1, and tyrosine phosphorylation of MAGI-1 determined. Similar results were observed with MAGI-2 (data not shown).

Figure 5

Position of the markers analyzed across the PTPRZ1 gene. Markers analyzed in phase 1 and phase 2 are shown, with markers with a nominal P > 0.05 indicated in red.