Targeting Human Epidermal Growth Factor Receptor Signaling with the Neuregulin's Heparin-binding Domain

Abstract

Neuregulin (NRG)2 has been shown to have a wide array of functions with important roles in nervous system and heart development as well as in diseases ranging from breast cancer to schizophrenia (1-4). It mediates its diverse effects through binding homo-and heterodimeric cell-surface epidermal growth factor receptors, including HER2(erbB2), HER3(erbB3), and HER4-(erbB4), that leads to rapid receptor-tyrosine phosphorylation and activation of downstream signaling pathways, including the mitogen-activated protein kinase and phosphatidylinositol 3-kinase (5). Many alternatively spliced forms of NRG are secreted after proteolytic cleavage from their transmembrane precursors (6). All of these forms have a receptor-binding epidermal growth factor-like domain together with a distinct C2 immunoglobulin-like domain that functions as a unique heparin-binding domain (HBD) with an alternating array of positively charged amino acids held together in a disulfide-bonded loop. This HBD binds specific sulfate groups on negatively charged heparan sulfate proteoglycans that leads to a highly specific tissue distribution of NRG (7-10).Because of its strong mitogenic effect in some breast cancers, blocking NRG signaling has become an attractive therapeutic target (11). Although a currently approved humanized monoclonal antibody against HER2 called trastuzumab (12) has become a clinically effective adjuvant therapy for a subgroup of breast cancer patients (13, 14), it does not effectively block HER2 activation induced by NRG autocrine and paracrine signaling (15-17). A major obstacle in blocking NRG signaling both in cancer and in developmental studies comes from its ability to become highly concentrated in the extracellular matrix where it can produce sustained HER activation required for downstream gene activations (7, 15). To overcome this obstacle, we have fused the human the HBD of NRG to the soluble ectodomain of HER4 (H4) with high affinity for epidermal growth factor-like domain as a means to target this NRG antagonist to the same heparan sulfate-rich cell surfaces that bind NRG. In this way, the antagonist could effectively compete with NRG on an even footing and would enable more effective studies of the role of NRG in development and in cancer. EXPERIMENTAL PROCEDURESConstruction of Fusion Proteins-All fusion proteins were derived entirely from human sequences. The extracellular domain of HER4 receptor (H4) corresponds to 99 -2042 bp of the human HER4 NM_005235 mRNA. The sequence from 99 -173 bp encodes a 25-amino acid signal sequence that was incorporated onto the N terminus of all constructs for secretion and protein expression. H4 was amplified by PCR and then inserted into pMH vector (Roche Applied Science, Indianapolis IN) between KpnI and EcoRI to generate the H4-HA construct. The HBD (532-849 bp) and HBD-S (532-1023 bp) domains of NRG ␤1 form (NM_013964) were amplified from the plasmid HARIA PATH 2 (gift of Dr. Tejvir Khurana, University of Pennsylvania). Either HBD-S or the HBD domain alone was inserted into pMH-H...

The Tanapoxvirus 15L Protein Is a Virus-Encoded Neuregulin That Promotes Viral Replication in Human Endothelial Cells

“…In fact, fusion proteins containing the Ig-like heparin binding domain from neuregulin retains heparin binding capacity, although at different affinities depending on the requirement of a spacer domain and where it fits into the context of the rest of the protein (26). Despite this, TPV-15L retains the functional capacity to bind heparin, which may be an indication of potential targeting and function of the molecule.…”

“…SPGF, on the other hand, has yet to be fully characterized, but some data suggest that ErbB1 is necessary for signal transduction (25). Here, we describe a fourth member of the EGF-like growth factor family from tanapoxvirus (TPV), encoded by the TPV-15L gene, that is capable of activating the neuregulin receptor heterodimer ErbB2/3 as shown in an established neuregulin bioassay (26). Our initial characterization of this protein has identified heparin binding ability, similar to the case for certain isoforms of mammalian neuregulin.…”

Complexity of trophic factor signaling in experimental autoimmune encephalomyelitis: Differential expression of neurotrophic and gliotrophic factors

“…However, a follow-up study indicated that effects of systemically delivered type II NRG1 were less likely due to direct effects on remyelination, but to modulation of the immune response that favors remyelination (Penderis et al, 2003). Given that microglial activation is a common pathological change in both ALS and MS, blocking NRG1 signaling could thus be a therapeutic target to slow disease progression in neurodegenerative diseases including MS. We have developed a targeted neuregulin antagonist (Ma et al, 2009) that given intrathecally reduces microglial activation in rat chronic spinal cord pain model (Calvo et al, 2010). Use of this new reagent could help define at which points during the disease process NRG1 activity is beneficial or harmful.…”

Aberrant Neuregulin 1 Signaling in Amyotrophic Lateral Sclerosis

“…Recently, NRG1 signaling through activation of erbB2 receptors has been shown to be critical for microglial activation in the dorsal spinal cord of rats that develop chronic pain following peripheral nerve injury (23). Using a novel NRG1 antagonist (45) injected intrathecally, the number of activated microglia was reduced together with mechanical and cold pain-related hypersensitivity (23). These findings suggest that NRG1 is an important, local signal for microglial activation in the spinal cord after neural injury and that blocking NRG1 signaling could be a novel approach to block the untoward effects that ensue.…”