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Neuregulins are a family of growth and differentiation factors that act through activation of cell-surface erbB receptor tyrosine kinases and have essential functions both during development and on the growth of cancer cells. One alternatively spliced neuregulin-1 form has a distinct heparin-binding immunoglobulin-like domain that enables it to adhere to heparan sulfate proteoglycans at key locations during development and substantially potentiates its activity. We examined the structural specificity needed for neuregulin-1-heparin interactions using a gel mobility shift assay together with an assay that measures the ability of specific oligosaccharides to block erbB receptor phosphorylation in L6 muscle cells. Whereas the N-sulfate group of heparin was most important, the 2-O-sulfate and 6-O-sulfate groups also contributed to neuregulin-1 binding in these two assays. Optimal binding to neuregulin-1 required eight or more heparin disaccharides; however, as few as two disaccharides were still able to bind neuregulin-1 to a lesser extent. The physiological importance of this specificity was shown both by chemical and siRNA treatment of cultured muscle cells. Pretreatment of muscle cells with chlorate that blocks all sulfation or with an siRNA that selectively blocks N-sulfation significantly reduced erbB receptor activation by neuregulin-1 but had no effect on the activity of neuregulin-1 that lacks the heparin-binding domain. These results suggest that the regulation of glycosaminoglycan sulfation is an important biological mechanism that can modulate both the localization and potentiation of neuregulin-1 signaling.

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...