Molecular and cellular characterization of Neuregulin-1 type IV isoforms

Abstract

Numerous genetic studies associated the Neuregulin 1 (NRG1) Icelandic haplotype (HAP(ice)), and its single nucleotide polymorphism SNP8NRG243177 [T/T], with schizophrenia. Because SNP8NRG243177 [T/T] has characteristics of a functional polymorphism that maps close to NRG1 type IV coding sequences, our initial goal was to map precisely the human type IV transcription initiation site. We determined that the initiation site is 23 bp upstream of the previously reported type IV exon, and that no other transcripts map to the SNP8NRG243177 region. Because NRG1 type IV transcripts are specific to human, we isolated full-length NRG1 type IV cDNAs from human hippocampi and expressed them in non-neural cells and dissociated rat hippocampal neurons to study protein expression, processing and function. Using an antiserum we generated against the NRG1 type IV-specific N-terminus, we found that the protein is targeted to the cell surface where PKC activation promotes its cleavage and release of the extracellular domain. Conditioned medium derived from type IV expressing cells stimulates ErbB receptor phosphorylation, as well as downstream Akt and Erk signaling, demonstrating that NRG1 type IV possesses biological activity similar to other releasable NRG1 isoforms. To study the subcellular targeting of distinct isoforms, neurons were transfected with the Ig-domain-containing NRG1 types I and IV, or the cysteine-rich domain type III isoform. Three dimensional confocal images from transfected neurons indicate that, whereas all isoforms are expressed on somato-dendritic membranes, only the type III-cysteine-rich domain isoform is detectable in distal axons. These results suggest that NRG1 type IV expression levels associated with SNP8NRG243177 [T/T] can selectively modify signaling of NRG1 released from somato-dendritic compartments, in contrast to the type III NRG1 that is also associated with axons.

Fig 1. Mapping the NRG1 type IV promoter and isolation of type IV transcripts

A) Genomic organization of NRG1 showing putative promoters and exons encoding distinct functional domains. The exons are represented by different color boxes, and putative promoters (exons I through VI) are indicated (gray boxes). Exons encoding the different domains are color-coded and represent: Ig-like domain (green), spacers (cyan), α and β EGF-like domains splice variants with extensions 1–4 (blue), TM (purple), cytoplasmic domains 1–3 (bourdeaux), and the distinct C-termini a and b (orange). Sites and orientations of primers used in this study are indicated by the arrows below. The sequence of the type IV exon is shown, including the novel upstream 23 bp sequence identified in this study (bold blue). Location of the 8NRGSNP243177 polymorphism is indicated. (B) Schematic representation of the human type IV isoforms encoded by the cDNAs isolated in this study (β1and β3). The domains contained in each protein, as defined in the diagram above, are shown. The unique 13 amino acid type IV human sequence used to generate a type IV specific antibody is shown below.

Fig 2. The NRG1 type IV antibody HL5792 identifies a glycosylated NRG1 type IVβ₁a protein

NRG1 type IV constructs expressing V5-tagged proteins were transfected into HEK293 cells. A) Western blots prepared with 10 μg protein from HEK293 whole cell extracts probed with HL5792 rabbit polyclonal antibody raised against the type IV N-terminus (left panel). A monoclonal antibody to the V5 epitope (middle) and a commercial antibody raised against the a-tail C-terminus (right) were used to confirm expression of full-length NRG1. Extracts from untransfected HEK293 cells were included as negative controls (Con). The 3 antibodies identified a major band at 90 kDa corresponding to pro-NRG1 type IV (arrow), while the C-terminal antibody also detected a putative processed ICD of ~ 65 kDa and additional truncated products (open arrows). The light bands observed at 75 and 50 kDa using HL5792 are nonspecific cross-reacting bands (open arrowheads), also seen in untransfected control. (B) Cell lysates were treated with O- or N-glycanase to determine extent of NRG1 type IV glycosylation. Western blots were probed with the V5 epitope tag antibody, stripped, and re-probed with anti-GAPDH. A shift in NRG1 type IV apparent molecular weight from 90 kD (open arrowhead, left) to 80 kD (closed arrowhead, right) was observed after N- and O- glycanase treatment; note that migration of GAPDH remains unchanged.

Fig 3. NRG1 type IV is targeted to the plasma membrane, proteolytically processed and released into the medium

(A) HEK-293 cells expressing a cDNA encoding NRG1 type IV tagged at its N-terminus (top panel) or C-terminus (bottom panel) with the V5 epitope were fixed, and incubated with either type IV- or V5-specific antibodies using non-permeabilizing conditions. Type IV protein is expressed at the cell surface. (B) Type IV ECD is released into the medium in the presence of phorbol ester. HEK293 cells expressing V5 tagged-type IV were incubated with or without 0.5 μM PMA for 30 min. The condition medium was collected and concentrated by filtration. Concentrated conditioned medium was probed by Western blotting with HL5792 and V5 antibodies. Both antibodies detect a 29 kDa band corresponding to the type IV ECD. (C) Type IV ECD concentrated medium treated with O- or N- Glycanase to the determine extent of NRG1 type IV glycosylation, and analyzed by Western blotting using anti-HL5792 antibodies. A downward shift in mobility of the deglycosylated type IV ECD was observed.

Fig 4. NRG1 type IV protein stimulates ErbB receptor and downstream signaling pathways

A) Induction of tyrosine phosphorylation by NRG β1 peptide and NRG1 isoforms. A dose-response curve of receptor autophosphorylation, assayed as an increase in phosphotyrosine signal levels at ~180 kDa upon stimulation with a EGF-like peptide (left panel). Conditioned medium form OVCAR-3 cell expressing different NRG1 isoforms was collected used to stimulate serum starved OVCAR-3 cells. Western blots of cell lysates were analyzed using the pan-phopho-tyrosine antibody. A 180 kDa band corresponding to pErbB was absent in parallel research with or without 10 μM PD158780 was included, (right panel). (B) Activation of pAkt and p42/44, downstream molecules of the NRG1/ErbB signaling pathway, by NRG1 isoforms. Immunoblot analysis was performed on total cell lysate using antibodies against phospho-Akt or phospho-Erk p44/42. Blots were striped and re-probe for total Akt and p44/p42 protein. (C) Quantitative analysis Western blots shown in B (Data mean+SD from 3 independent experiments).

Fig 5. Subcellular localization of NRG1 type IV in hippocampal neurons

A) Dissociated hippocampal neurons co-transfected with NRG1 type IV and SNPH-GFP, and live labeled with type IV antibodies. Cells were also stained for the dendritic marker MAP2 after permeabilization. Type IV immunolabeling (red) colocalizes with the dendritic marker MAP2 (blue) and is strongest in the proximal region of dendrites. (B) Hippocampal neurons were co-transfected with NRG1 type I, type IV or type III and SNPH-GFP (green), permeabilized, and incubated with antibodies against NRG1 (red) and MAP2 (blue). Type I and IV immunoreactivity is restricted to neuronal somas and dendrites, and absent from SNPH positive axons. In contrast, NRG1 CRD-type III is localized to the cell somata and dendrites but it also was targeted to the axon (green). (C) Three dimension reconstruction of the area obtained by the dash box in B. Axons (green) are indicated by black arrows. Type IV and I immunolabeling can be observed in the proximal region of the axon (white arrowheads) but not in more distal region. Unlike type IV and I, CRD-type III is expressed in the axon (white filled arrowheads).