N-arginine dibasic convertase is a specific receptor for heparin-binding EGF-like growth factor that mediates cell migration

Abstract

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a mitogen and chemotactic factor, binds to two receptor tyrosine kinases, erbB1 and erbB4. Now we demonstrate that HB-EGF also binds to a novel 140 kDa receptor on MDA-MB 453 cells. Purification of this receptor showed it to be identical to N-arginine dibasic convertase (NRDc), a metalloendopeptidase of the M16 family. Binding to cell surface NRDc and NRDc in solution was highly specific for HB-EGF among EGF family members. When overexpressed in cells, NRDc enhanced their migration in response to HB-EGF but not to EGF. Conversely, inhibition of endogenous NRDc expression in cells by antisense morpholino oligonucleotides inhibited HB-EGF-induced cell migration. Anti-erbB1 neutralizing antibodies completely abrogated the ability of NRDc to enhance HB-EGF-dependent migration, demonstrating that this NRDc activity was dependent on erbB1 signaling. Although NRDc is a metalloproteinase, enzymatic activity was not required for HB-EGF binding or enhancement of cell migration; neither did NRDc cleave HB-EGF. Together, these results suggest that NRDc is a novel specific receptor for HB-EGF that modulates HB-EGF-induced cell migration via erbB1.

Fig. 1. Cross-linking of HB-EGF to 453 cells. (A) [¹²⁵I]HB-EGF (5 ng/ml) was cross-linked to 3T3/erbB1 cells (lane 1) and 453 cells (lane 2). [¹²⁵I]EGF (lane 3), [¹²⁵I]BTC (lane 4) and [¹²⁵I]HRGβ (lane 5) (5 ng/ml each) were bound and cross-linked to 453 cells in 6 cm dishes. The cells were lysed, and proteins were resolved by 6% SDS–PAGE and visualized by autoradiography. (B) [¹²⁵I]HB-EGF (5 ng/ml) was cross-linked to 453 cells in the presence of 1.25 µg/ml unlabeled HB-EGF, EGF, AR, BTC or HRGβ in 6 cm dishes. The cells were lysed, and proteins were resolved by 6% SDS–PAGE followed by autoradiography as described in (A). The arrows point to the 150 kDa radiolabeled complex containing [¹²⁵I]HB-EGF and HB-EGF receptor.

Fig. 2. Purification of HB-EGF receptor from 453 cells. (A) Upper panel: [¹²⁵I]HB-EGF was cross-linked to MonoQ column fractions in solution. Lower panel: aliquots of column fractions were analyzed by SDS–PAGE and Coomassie Blue staining. ST is an aliquot of the starting material applied to the MonoQ column, which had been partially purified by SuperQ and hydroxyapatite chromatography. The arrow in the lower panel indicates the band in fraction 22 used for N-terminal protein sequencing. (B) The first 70 amino acids of NRDc are shown. The nine-amino-acid N-terminal amino acid sequence determined by microsequencing is depicted in bold and begins at NRDc amino acid 51.

Fig. 3. Immunochemical and in vitro translation studies suggest that the HB-EGF specific receptor is NRDc. (A) [¹²⁵I]HB-EGF was cross-linked in solution to an aliquot of fraction 21 shown in Figure 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 by 6% SDS–PAGE, 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 in vitro translation of NRDc mRNA. [¹²⁵I]HB-EGF cross-linking to HB-EGF receptor [the starting material (ST) in Figure 2A, designated here as HB-EGFR] is shown in lane 1 for comparison. The arrow denotes radiolabeled complexes containing [¹²⁵I]HB-EGF and NRDc.

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 antibody (lane 7). Lane 5, total cell lysates prior to immuno precipitation. Immunocomplexes were resolved by 6% SDS–PAGE, followed by autoradiography. Open and solid arrows denote radiolabeled complexes containing [¹²⁵I]HB-EGF and erbB1, and [¹²⁵I]HB-EGF and NRDc, respectively.

Fig. 5. Characterization of HB-EGF–NRDc interactions. (A) Scatchard analysis of HB-EGF binding to NRDc. Left panel: increasing amounts of [¹²⁵I]HB-EGF (0.78–200 ng/ml) were added to partially purified rat NRDc in solution, followed by immunoprecipitation with anti-NRDc antibody. Immunoprecipitated complex-associated radioactivity was determined by a gamma counter. Non-specific binding was determined by competition with a 200-fold excess of unlabeled HB-EGF. Right panel: the binding data shown in the left panel were analyzed by the method of Scatchard, and best-fit plots were obtained using the LIGAND program. (B) NRDc ligand-binding specificity in solution. [¹²⁵I]HB-EGF (47 ng/ml) was bound and cross-linked to recombinant rat NRDc in the presence of 47 ng/ml (lane 2), 470 ng/ml (lane 3), 4.7 µg/ml (lane 4) unlabeled HB-EGF, or 4.7 µg/ml unlabeled EGF (lane 5), TGF-α (lane 6), AR (lane 7), BTC (lane 8), HRGβ (lane 9), bFGF (lane 10), or VEGF₁₆₅ (lane 11). The arrow denotes radiolabeled complexes containing [¹²⁵I]HB-EGF and NRDc.

Fig. 6. Expression of NRDc in HeLa cells enhances HB-EGF-induced migration via erbB1. (A) The migration of HeLa cells transiently transfected with control vector (open squares) or NRDc expression vector (closed squares) in response to HB-EGF (left panel) or EGF (right panel) was measured in a Boyden chamber. Each point represents the mean and standard error of eight (HB-EGF) or six (EGF) independent experiments. Each experiment was carried out in quadruplicate. *P <0.05. (B) Inhibition of NRDc-enhanced cell migration by an anti-erbB1 blocking antibody. HeLa cells transiently transfected with control vector (lanes 1–4) or NRDc expression vector (lanes 5–8) were pre-treated with (gray bar; lanes 2, 4, 6, 8), or without (black bar; lanes 1, 3, 5, 7) 10 µg/ml anti-erbB1 blocking antibody (C225), and then incubated with or without 10 ng/ml HB-EGF in Boyden chamber migration assays. Each bar represents the mean and standard error of four independent experiments, normalized to the value for migrated cell numbers of control vector-transfected cells without C225 treatment, and without HB-EGF stimulation. Each experiment was carried out in quadruplicate.

Fig. 7. Antisense morpholino oligonucleotides inhibit NRDc protein synthesis and inhibit HB-EGF-induced migration. (A) HaCaT keratinocytes were treated with a 1.1 µM concentration of either standard morpholino oligonucleotides (control) or NRDc antisense morpholino oligonucleotides (AS) for 48 h. Total cell lysates (140 µg) of oligonucleotide-treated cells were analyzed by western blotting with anti-NRDc antibody. The same membrane was stripped and re-blotted with anti-erbB1 antibody. (B) The intensity of signals in (A) was quantitated by densitometry using the NIH image program. The relative densities of erbB1 (lanes 1 and 2) and NRDc protein levels (lanes 3 and 4) in antisense oligonucleotide-treated cells (lanes 2 and 4) compared to control oligonucleotide-treated cells (lanes 1 and 3) are shown. (C) The migration of HaCaT keratinocytes treated with standard control oligonucleotides (lane 1) or NRDc antisense oligonucleotides (lane 2) in response to HB-EGF (10 ng/ml) was measured in a Boyden chamber. Each value represents the mean and standard error of eight samples from a single experiment, and is representative of the same qualitative effect seen in two such experiments.

Fig. 8. NRDc enzymatic activity is not needed for NRDc binding to HB-EGF or NRDc-induced migration. (A) HB-EGF binds to enzymatically inactive NRDc. [¹²⁵I]HB-EGF (5 ng/ml) was bound and cross-linked to HeLa cells transiently transfected with control vector (lane 1), a vector expressing wild-type rat NRDc (WT; lane 2) or a vector expressing enzymatically inactive mutant of NRDc (Mutant; lane 3). The arrow denotes radiolabeled complexes containing [¹²⁵I]HB-EGF and NRDc. (B) Enzymatically inactive NRDc enhances HB-EGF-induced cell migration. Migration of HeLa cells transiently transfected with control vector (open squares), NRDC expression vector (closed squares) or an NRDC expression vector expressing enzymatically inactive (mutant) NRDc (open circles) in response to HB-EGF was measured in a Boyden chamber. Cells were lysed and analyzed by western blotting with anti-NRDc antibody (inset). Each point represents the mean cell number and standard deviations of four different wells from a single experiment, and is representative of the same qualitative effect seen in three such experiments.

Fig. 9. NRDc does not cleave HB-EGF. (A) Cell surface HB-EGF: An expression plasmid encoding AP-transmembrane HB-EGF was co-transfected into HeLa cells with either a control vector (lanes 1 and 2) or an NRDc expression vector (lane 3). After a 24 h incubation, the control vector-transfected cells were treated without (lane 1) or with (lane 2) 100 nM PMA for 30 min. Conditioned medium (CM) of control cells, control cells treated with PMA, or cells expressing NRDc were assayed for relative AP activity compared with control, arbitrarily set as 1. Each bar is the average of triplicate values. (B) HB-EGF in solution. [¹²⁵I]HB-EGF (60 ng/ml) was incubated with no (lane 1), 100 ng/ml (lane 2) or 250 ng/ml (lane 3) wild-type NRDc in solution for 1 h at 37°C. [¹²⁵I]HB-EGF size was analyzed on a 15% SDS–PAGE, followed by autoradiography.