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. 1997 Sep 2;94(18):9562-7.
doi: 10.1073/pnas.94.18.9562.

Neuregulin-3 (NRG3): A Novel Neural Tissue-Enriched Protein That Binds and Activates ErbB4

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Free PMC article

Neuregulin-3 (NRG3): A Novel Neural Tissue-Enriched Protein That Binds and Activates ErbB4

D Zhang et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

We describe the identification of Neuregulin-3 (NRG3), a novel protein that is structurally related to the neuregulins (NRG1). The NRG1/neuregulins are a diverse family of proteins that arise by alternative splicing from a single gene. These proteins play an important role in controlling the growth and differentiation of glial, epithelial, and muscle cells. The biological effects of NRG1 are mediated by receptor tyrosine kinases ErbB2, ErbB3, and ErbB4. However, genetic studies have suggested that the activity of ErbB4 may also be regulated in the central nervous system by a ligand distinct from NRG1. NRG3 is predicted to contain an extracellular domain with an epidermal growth factor (EGF) motif, a transmembrane domain, and a large cytoplasmic domain. We show that the EGF-like domain of NRG3 binds to the extracellular domain of ErbB4 in vitro. Moreover, NRG3 binds to ErbB4 expressed on cells and stimulates tyrosine phosphorylation of this receptor. The expression of NRG3 is highly restricted to the developing and adult nervous system. These data suggest that NRG3 is a novel, neural-enriched ligand for ErbB4.

Figures

Figure 1
Figure 1
cDNA sequence analysis of NRG3. (A) Amino acid sequences of human and murine NRG3 (hNRG3 and mNRG3, respectively). The EGF-like domain, the N-terminal hydrophobic segment (double underline), the serine/threonine-rich portion, and a predicted transmembrane domain (single underline) are highlighted. (B) Sequence alignment analysis for the EGF-like domains of human NRG3 (hNRG3), chicken ARIA (cARIA) (4), human amphiregulin (hAR) (18), human betacellulin (hBTC) (19), human EGF (hEGF) (20), human heparin-binding EGF-like growth factor (hHB-EGF) (21), human neuregulin-1α (hNRG1α), human neuregulin-1β (hNRG1β) (2), mouse NRG2 (mNRG2) (12, 13), human TGF-α (hTGF-α) (43), and mouse epiregulin (mEPR) (22). The sequences were analyzed using Sequence Analysis Programs (Genentech).
Figure 2
Figure 2
Multi-tissue Northern blot analysis of NRG3. (A) Northern blot analysis of mRNA from human tissues hybridized to a human NRG3 specific probe. RNA size markers (in kb) are shown on the left of each blot. Lanes 1–16 represent poly(A)+ mRNA from lanes: 1, heart; 2, brain; 3, placenta; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; 8, pancreas; 9, spleen; 10, thymus; 11, prostate; 12, testis; 13, ovary; 14, small intestine; 15, colon (mucosal lining); 16, peripheral blood leukocytes. (B) Northern blot analysis of mRNA from human brain tissues using the same probe as in A. Lanes 1–8 represent poly(A)+ mRNA from lanes: 1, amygdala; 2, caudate nucleus; 3, corpus callosum; 4, hippocampus; 5, whole brain; 6, substantia nigra; 7, subthalamic nucleus; 8, thalamus. Northern blot analysis using mRNA from multiple murine tissues and murine NRG3 cDNA probe also revealed a 4.4 kb signal with a similar expression pattern as human NRG3 (not shown). β-actin signal was used for loading control.
Figure 3
Figure 3
In situ hybridization analysis of NRG3 RNA expression. (A) Low-power brightfield view of an E16 embryo section stained with hematoxylin and eosin. Structures labeled by the NRG3 probe are identified: sc, spinal cord; sg, spinal ganglion; t, trigeminal ganglion; vc; vestibular-cochlear ganglion; cp, cortical plate. (B) Darkfield view of the same section demonstrating the pattern of NRG3 hybridization. Intense hybridization signal appears exclusively over neural tissue. In the developing cerebral cortex, NRG3 signal is detected in the cortical plate, but not in underlying regions. (C) Darkfield view of an adjacent section hybridized with the NRG3 sense control probe. (D Upper) A sagittal section of formalin-fixed adult mouse brain hybridized with NRG3 sense probe observed under darkfield illumination. (Lower) An adjacent section hybridized with NRG3 antisense probe and viewed under darkfield illumination. Note the strong signal in cerebral cortex, hippocampus, and thalamus. CTX, cerebral cortex; HC, hippocampus; TH, thalamus.
Figure 4
Figure 4
Binding of NRG3EGF.Fc to ErbB4. (A) Binding of NRG3EGF.Fc to K562erbB cells as analyzed by FACS. (Panels 1-4) Parental K562 cells (1) or K562 cells expressing either ErbB2 (K562erbB2 cells, 2), ErbB3 (K562erbB3 cells, 3), or ErbB4 (K562erbB4 cells, 4) were examined for the expression of corresponding receptors. Cells were incubated with anti-ErbB2, anti-ErbB3, or anti-ErbB4 antibodies as indicated before the phycoerythrin-conjugated secondary antibody was added. The binding of antibodies was analyzed by FACS. LOG PE, relative fluorescent intensity; Counts, cell numbers. (Panels 5-8) NRG3EGF.Fc binds to ErbB4 expressing cells. Parental K562 cells (5), K562erbB2 cells (6), K562erbB3 cells (7), and K562erbB4 cells (8) were incubated with or without NRG3EGF.Fc (containing gD tag) for 1 hr, followed by anti-gD-tag primary antibody and phycoerythrin-conjugated secondary antibody. The binding of antibodies was evaluated by FACS analysis. (B) Competitive inhibition of 125I-NRG3EGF.Fc binding to immobilized ErbB4.Fc by NRG3EGF.Fc or NRG1EGF. Iodination of NRG3EGF.Fc and the binding assay were as described in Materials and Methods. ErbB4.Fc was immobilized on 96-well plates and incubated with various concentrations of unlabeled NRG3EGF.Fc or NRG1EGF and a constant amount of 125I-labeled NRG3EGF.Fc for 1.5 hr at room temperature. Unbound ligand was removed and the plate was extensively washed. The fraction of radioactivity bound over total 125I-NRG3EGF.Fc input is plotted against the concentration of competitor. Data of a representative experiment from four independent assays are shown. Error bars indicate standard deviation of quadruplicate samples of this experiment.
Figure 5
Figure 5
Tyrosine phosphorylation of transfected and endogenous ErbB4 by NRG3EGF.Fc. (A) K562erbB4 cells were untreated (lane 1), or treated with 2.5 nM (lanes 2 and 4) and 25 nM (lanes 3 and 5) of NRG3EGF.Fc for 3 min (lanes 2 and 3) and 8 min (lanes 4 and 5). The cells were lysed and immunoprecipitated by anti-ErbB4 antibody, and probed with peroxidase-conjugated anti-phosphotyrosine antibody (Transduction Laboratories). *, a truncated ErbB4. The Mr (in kDa) is indicated at left. (B) MDA-MB-453 cells were untreated (lane 1) or treated with 2 nM, 20 nM, or 200 nM of NRG3EGF.Fc (lanes 2, 3, and 4, respectively), with 2 nM, 20 nM, or 200 nM of NRG3EGF.H6 (lanes 5, 6, and 7, respectively) or with 20 nM NRG1EGF (lane 8) for 3 min. The cells were lysed and subjected to immunoprecipitation and immunoblot as described in A. The Mr (in kDa) is indicated at left. For the blots shown in A and B, equal loading was confirmed by reprobing with anti-ErbB4 antibody (not shown).

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