Total cell lysates (140?g) of oligonucleotide-treated cells were analyzed by western blotting with anti-NRDc antibody. dependent on erbB1 signaling. Although NRDc is usually a metalloproteinase, enzymatic activity was not required for HB-EGF binding or enhancement of Amylin (rat) cell migration; neither did NRDc cleave HB-EGF. Together, these results suggest that NRDc is usually a novel specific receptor for HB-EGF that modulates HB-EGF-induced cell migration via erbB1. and studies, HB-EGF has been implicated as a contributor to wound healing (Marikovsky et al., 1993), blastocyst implantation (Das et al., 1994), SMC hyperplasia as occurs in artherosclerosis (Miyagawa et al., 1995; Nishi et al., 1997) and pulmonary hypertension (Powell et al., 1993), the response to brain injury (Opanashuk et al., 1999; Tanaka et al., 1999), and oncogenic transformation (Fu et al., 1999). HB-EGF activity is usually mediated by high-affinity EGF-receptor tyrosine kinases. Four related receptor tyrosine kinases (RTKs) have been identified (reviewed by Hynes and Stern, 1994; Riese and Stern, 1998; Olayioye toxin fusion proteins (HB-EGFCPE) on mouse blastocysts, it was observed that HB-EGFCPE, but not TGF-CPE, was toxic for egfrC/C null (lacking erbB1) blastocysts, suggesting that a receptor other than erbB1 was a target for HB-EGF (Paria et al., 1999). [125I]HB-EGF cross-linking to mouse blastocysts resulted in the formation of 150C160?kDa complexes, which were smaller than the 180C190?kDa complexes formed by HB-EGF with erbB1 and erbB4 (Elenius et Rabbit polyclonal to PPA1 al., 1997c). Complexes of 150C160?kDa were also detected in human breast cancer MDA-MB?453, a cell line that expresses little erbB1 or erbB4 (Elenius et al., 1997c). Given these results, our goal was to purify a possible novel HB-EGF receptor on MDA-MB?453 cells. In this report, we describe the purification and characterization of this HB-EGF receptor, and show it to be identical to an N-terminal-truncated form of N-arginine (R) dibasic convertase (NRDc; nardilysin; EC 3.4.24.61), a member of the M16 family of metalloendopeptidases (Chesneau transcription and translation from NRDc cDNA, resulted in the formation of a 150?kDa complex (Physique?3B, lane 3) that co-migrated with a complex of [125I]HB-EGF and HB-EGF receptor partially Amylin (rat) purified from 453 cells (Physique?3B, lane 1). Open in a separate window Fig. 3. Immunochemical and translation studies suggest that the HB-EGF specific receptor is usually NRDc. (A)?[125I]HB-EGF was cross-linked in solution to an aliquot of fraction 21 shown in Physique?2A, yielding a 150?kDa complex (lane 1). This complex (bracket) was immuno precipitated with control IgG (lane 2) or anti-NRDc antibody (lane 3). The immunocomplexes were resolved Amylin (rat) by 6% SDSCPAGE, followed by autoradiography. (B)?[125I]HB-EGF was cross-linked in solution in the absence (lane 2) or presence (lane 3) of recombinant NRDc (rNRDc) prepared by translation of NRDc mRNA. [125I]HB-EGF cross-linking to HB-EGF receptor [the starting material (ST) in Physique?2A, designated here as HB-EGFR] is shown in lane 1 for comparison. The arrow denotes radiolabeled complexes made up of [125I]HB-EGF and NRDc. HeLa cells, which express erbB1?but very Amylin (rat) low levels of NRDc, were transiently transfected with NRDc cDNA. [125I]HB-EGF formed 180 and 150?kDa complexes with HeLa cells transiently expressing NRDc (Physique?4, lane 2), but only a 180?kDa complex in HeLa cells transfected with control vector (Physique?4, lane 1). [125I]EGF formed only 180?kDa complexes in HeLa cells whether they expressed (Physique?4, lane 4) or did not express NRDc (Physique?4, lane 3), evidence that this 180?kDa complex Amylin (rat) contains erbB1 and that EGF does not bind to cell surface NRDc. Immunoprecipitation with anti-erbB1 antibody (Physique?4, lane 6) or anti-NRDc (Physique?4, lane 7) confirmed that this 180?kDa complex contained erbB1 and the 150?kDa complex contained NRDc, respectively. Open in a separate window Fig. 4. NRDc transiently expressed in HeLa cells binds [125I]HB-EGF. Left: [125I]HB-EGF (5?ng/ml) (lanes 1 and 2) or [125I]EGF (5?ng/ml) (lanes 3 and 4) was cross-linked to HeLa cells transiently transfected with control vector (lanes 1and 3) or NRDc expression vector (lanes 2 and 4). Right: after [125I]HB-EGF cross-linking to HeLa cells transiently transfected with NRDc expression vector, total cell lysates were immunoprecipitated with anti-erbB1 antibody (lane 6) or anti-NRDc.