5232 • The Journal of Neuroscience, May 24, 2017 • 37(21):5232–5249 Cellular/Molecular Structural Similarities between Neuregulin 1–3 Isoforms Determine Their Subcellular Distribution and Signaling Mode in Central Neurons Detlef Vullhorst,* Tanveer Ahmad,* Irina Karavanova, Carolyn Keating, and Andres Buonanno Section on Molecular Neurobiology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland 20892 The Neuregulin (NRG) family of ErbB ligands is comprised of numerous variants originating from the use of different genes, alternative promoters, and splice variants. NRGs have generally been thought to be transported to axons and presynaptic terminals where they signal via ErbB3/4 receptors in paracrine or juxtacrine mode. However, we recently demonstrated that unprocessed pro-NRG2 accumulates on cell bodies and proximal dendrites, and that NMDAR activity is required for shedding of its ectodomain by metalloproteinases. Here we systematicallyinvestigatedthesubcellulardistributionandprocessingofmajorNRGisoformsinrathippocampalneurons.Weshowthat NRG1 isotypes I and II, which like NRG2 are single-pass transmembrane proteins with an Ig-like domain, share the same subcellular distribution and ectodomain shedding properties. We furthermore show that NRG3, like CRD-NRG1, is a dual-pass transmembrane protein that harbors a second transmembrane domain near its amino terminus. Both NRG3 and CRD-NRG1 cluster on axons through juxtacrine interactions with ErbB4 present on GABAergic interneurons. Interestingly, although single-pass NRGs accumulate as unpro- cessed proforms, axonal puncta of CRD-NRG1 and NRG3 are comprised of processed protein. Mutations of CRD-NRG1 and NRG3 that render them resistant to BACE cleavage, as well as BACE inhibition, result in the loss of axonal puncta and in the accumulation of unprocessed proforms in neuronal soma. Together, these results define two groups of NRGs with distinct membrane topologies and fundamentally different targeting and processing properties in central neurons. The implications of this functional diversity for the regulation of neuronal processes by the NRG/ErbB pathway are discussed. Key words: BACE; ErbB4; metalloproteinases; Neuregulin; NMDA receptor; processing Significance Statement Numerous Neuregulins (NRGs) are generated through the use of different genes, promoters, and alternative splicing, but the functional significance of this evolutionary conserved diversity remains poorly understood. Here we show that NRGs can be categorized by their membrane topologies. Single-pass NRGs, such as NRG1 Types I/II and NRG2, accumulate as unprocessed proforms on cell bodies, and their ectodomains are shed by metalloproteinases in response to NMDA receptor activation. By contrast, dual-pass CRD-NRG1 and NRG3 are constitutively processed by BACE and accumulate on axons where they interact with ErbB4 in juxtacrine mode. These findings reveal a previously unknown functional relationship between membrane topology, protein processing, and subcellular distribution, and suggest that single- and dual-pass NRGs regulate neuronal functions in fundamentally different ways. Introduction processes (Buonanno, 2010; Mei and Nave, 2014). For example, Neuregulins (NRGs) signal via ErbB3/4 receptor tyrosine kinases NRG/ErbB4 signaling in parvalbumin-positive GABAergic in- to regulate numerous neurodevelopmental and activity-dependent terneurons regulates glutamatergic synaptic plasticity in the hip- Received Aug. 19, 2016; revised March 14, 2017; accepted April 9, 2017. The authors declare no competing financial interests. Authorcontributions:D.V.,T.A.,andA.B.designedresearch;D.V.,T.A.,I.K.,andC.K.performedresearch;D.V.and *D.V. and T.A. contributed equally to this study. T.A. contributed unpublished reagents/analytic tools; D.V., T.A., and A.B. analyzed data; D.V., T.A., and A.B. wrote Correspondence should be addressed to Dr. Andres Buonanno, Eunice Kennedy Shriver National Institute the paper. of Child Health and Human Development, 35 Lincoln Drive, Room 2C-1000, Bethesda, MD 20892. E-mail: This work was supported by Eunice Kennedy Shriver Institute of Child Health and Human Development Intramu- [email protected]. ral Research Program. We thank Drs. Vincent Schram and Carolyn Smith (Porter Neuroscience Center imaging core) C. Keating’s present address: Department of Neuroscience, University of Pennsylvania, Philadelphia, PA 19104. for expert assistance with confocal microscopy; and Dr. Jung-Hwa Tao-Cheng (National Institute of Neurological DOI:10.1523/JNEUROSCI.2630-16.2017 Disorders and Stroke EM facility) for help with immunogold EM microscopy. Copyright © 2017 the authors 0270-6474/17/375232-18$15.00/0 Vullhorst, Ahmad et al. • Subcellular Neuregulin Targeting in Central Neurons J. Neurosci., May 24, 2017 • 37(21):5232–5249 • 5233 pocampus (Chen et al., 2010; Shamir et al., 2012) and critical In this study, we delineated general principles underlying the period plasticity in the visual cortex (Gu et al., 2016; Sun et al., relationship between domain structure, subcellular distribution, 2016). NRGs are encoded by four genes, of which nrg1–3 are and ectodomain shedding of major NRG isoforms in central neu- expressed in the nervous system. All NRGs share an EGF-like rons, including NRG1 Types I/II, NRG2, and NRG3. Our data domain that mediates receptor binding and initiation of down- show that single-pass NRGs share the same subcellular targeting stream signaling. NRG1 isoform diversity results from the use of and NMDAR-dependent ectodomain shedding properties, whereas multiple promoters that give rise to unique amino-terminal the dual-pass proteins CRD-NRG1 and NRG3 accumulate as sequences of NRG Types I–VI, and by alternative splicing of se- processed proteins on axons through juxtracrine interactions quences encoding both the extracellular and intracellular do- with ErbB4. Furthermore, processing of single-pass NRGs is me- mains (Falls, 2003; Steinthorsdottir et al., 2004). Proforms of diated by metalloproteinases, whereas dual-pass NRGs are pro- canonical NRGs, such as NRG1 Types I/II and NRG2, harbor a cessed by BACE. Together, these findings establish a rational single transmembrane pass (TMC) downstream of the EGF-like basis to understand NRG isoform diversity and function in cell motif that separates the amino-terminal extracellular domain biological terms. (ECD) from the carboxyl-terminal intracellular domain (ICD). They additionally contain an immunoglobulin-like (Ig) motif that mediates interactions with heparan sulfate proteoglycans in Materials and Methods the extracellular matrix (Loeb and Fischbach, 1995). Ig-NRGs Animals. Sprague Dawley rats of either sex were used for tissue culture shed their ectodomains upon proteolytic processing near the and RNA analysis. Wild-type C57BL/6J mice of either sex were used for immunohistochemistry and ISH. Animals were treated in accordance TMC, which then interact with ErbB receptors in paracrine man- with National Institutes of Health Animal Welfare guidelines. All proce- ner. By contrast, NRG1 isoforms transcribed from the Type III dures were approved by the National Institute of Child Health and promoter lack the Ig-like motif and instead contain a cysteine- Human Development Animal Care and User Committee. rich domain (CRD) near their amino terminus that serves as a Antibodies. Antibodies included the following: rabbit monoclonal second transmembrane domain (TMN). As a result, CRD-NRG1 mAB11 against NRG2 (Vullhorst et al., 2015); mouse monoclonal remains cell-attached after cleavage and signals in juxtacrine against Ankyrin G (clone N106/65; NeuroMab); rabbit polyclonal cus- fashion (Wang et al., 2001). NRG3, whose mode of signaling has tom antibody 5721 against Erbb4 (for immunization strategy, see Vull- not been reported, lacks both Ig-like and CRD motifs but in- horst et al., 2009); mouse monoclonal against Kv2.1 (clone K89/34; cludes a stretch of hydrophobic residues near its amino terminus NeuroMab); rabbit polyclonal against NRG1 (SC-348; Santa Cruz Bio- (Zhang et al., 1997). technology); mouse monoclonal against V5 tag (clone SV-Pk1; Bio- Rad); mouse monoclonal against HA tag (clone 16B12; Abcam); mouse Historically, the relationship between domain organization, monoclonal against PSD95 (clone 7E3-1B8; Thermo Fisher); rat signaling mode, and biological function has been most exten- monoclonal against GFP (clone GF090R; Nacalai Tesque); guinea pig sively explored for CRD-NRG1. In particular, juxtacrine interac- polyclonal against parvalbumin (Synaptic Systems); and guinea pig tions between CRD-NRG1 on sensory neuron or motorneuron polyclonal against vesicular acetylcholine transporter (vAChT; Syn- axons and ErbB2/3 receptors on glial cells are essential for Schwann aptic Systems). cell development and functional maturation (Garratt et al., 2000; Generation of an attenuated CMV promoter to drive low-level NRG Nave and Salzer, 2006), and mice lacking this isoform exhibit expression in transfected hippocampal neurons. To attain low to moderate deficient myelination of peripheral nerves resulting in neonatal NRG expression levels in transfected cultured neurons, the 586 bp wild- lethality (Wolpowitz et al., 2000). Axonal accumulation of CRD- type CMV promoter was “gutted” to lower its transcriptional output, NRG1 has therefore served as a general paradigm for subcellular following a previously described strategy (Watanabe and Mitchison, 2002). Briefly, a 434 bp internal fragment was removed from the