Molecular dissection of the human alpha2-macroglobulin subunit reveals domains with antagonistic activities in cell signaling - PubMed (original) (raw)

Molecular dissection of the human alpha2-macroglobulin subunit reveals domains with antagonistic activities in cell signaling

Elisabetta Mantuano et al. J Biol Chem. 2008.

Abstract

alpha(2)-Macroglobulin (alpha(2)M) is a plasma protease inhibitor, which reversibly binds growth factors and, in its activated form, binds to low density lipoprotein receptor-related protein (LRP-1), an endocytic receptor with cell signaling activity. Because distinct domains in alpha(2)M are responsible for its various functions, we hypothesized that the overall effects of alpha(2)M on cell physiology reflect the integrated activities of multiple domains, some of which may be antagonistic. To test this hypothesis, we expressed the growth factor carrier site and the LRP-1 recognition domain (RBD) as separate GST fusion proteins (FP3 and FP6, respectively). FP6 rapidly and robustly activated Akt and ERK/MAP kinase in Schwann cells and PC12 cells. This response was blocked by LRP-1 gene silencing or by co-incubation with the LRP-1 antagonist, receptor-associated protein. The activity of FP6 also was blocked by mutating Lys(1370) and Lys(1374), which precludes LRP-1 binding. FP3 blocked activation of Akt and ERK/MAP kinase in response to nerve growth factor-beta (NGF-beta) but not FP6. In PC12 cells, FP6 promoted neurite outgrowth and expression of growth-associated protein-43, whereas FP3 antagonized the same responses when NGF-beta was added. The ability of FP6 to trigger LRP-1-dependent cell signaling in PC12 cells was reproduced by the 18-kDa RBD, isolated from plasma-purified alpha(2)M by proteolysis and chromatography. We propose that the effects of intact alpha(2)M on cell physiology reflect the degree of penetration of activities associated with different domains, such as FP3 and FP6, which may be regulated asynchronously by conformational change and by other regulatory proteins in the cellular microenvironment.

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Figures

FIGURE 1.

FIGURE 1.

Sequences of α2M peptide-GST fusion proteins. The amino acid numbering is based on the mature α2M subunit. The fusion protein FP3-(591–774) includes the characterized binding site for growth factors. FP4 has no known activity. FP6 includes the α-helix and two Lys residues (Lys1370 and Lys1374), which are essential for LRP-1 binding. In FP6(K → A), Lys1370 and Lys1374 are mutated to Ala. MG6 and MG8 are predicted domains within the structure of α2M, which contain the growth factor-binding site and the RBD, respectively.

FIGURE 2.

FIGURE 2.

Specific binding of FP6 to LRP-1. A, LRP-1-expressing PEA-10 cells and LRP-1-deficient MEF-2 cells were incubated with 5 n

m

125I-FP6 for 4 h at 4 °C, in the presence and absence of non-radiolabeled FP6 (50 n

m

) or 0.2 μ

m

GST-RAP. Cell-associated radioactivity was determined. The results show the mean ± S.D., n = 3. B, FP6 was treated with 0.5 μ

m

trypsin for the indicated times. The trypsin was rapidly inactivated with pNPGB, and the sample subjected to SDS-PAGE and Coomassie Blue staining. The time 0 point was treated with pNPGB prior to trypsin. The lane labeled C shows the initial preparation of FP6. The_arrow_ shows the band at 24 kDa, referred to in the text.

FIGURE 3.

FIGURE 3.

FP6 activates Akt and ERK/MAP kinase in Schwann cells and PC12 cells. Schwann cells (A) and PC12 cells (B) were treated with vehicle (serum-free medium, SFM), α2M-MA (50 n

m

) or with increasing concentrations of FP6 (50–200 n

m

) for 10 min as indicated. Schwann cells also were treated with PDGF-BB (0.1 μg/ml) or Epo (1 n

m

) as positive controls and with 50 n

m

FP4 as a negative control. PC12 cells were treated with NGF-β (50 ng/ml) or 50 n

m

FP4. Protein extracts were subjected to SDS-PAGE and immunoblot analysis to detect phosphorylated Akt (pAKT), phosphorylated ERK/MAP kinase (pERK), total ERK/MAP kinase, total Akt, and β-actin. The blots shown are representative of three independent studies.

FIGURE 4.

FIGURE 4.

Activation of cell signaling by FP6 requires LRP-1. Schwann cells (A) and PC12 cells (B) were treated with vehicle (serum-free medium, SFM), α2M-MA (50 n

m

), FP6 (100 n

m

), FP6(K → A) (100 n

m

), or FP4 (100 n

m

) for 10 min. PDGF-BB, Epo, or NGF-β were added as positive controls. Protein extracts were subjected to immunoblot analysis to detect pAKT, pERK, and total ERK/MAP kinase (total ERK). Schwann cells (C) and PC12 cells (D) were treated with FP6 (50 or 100 n

m

), Epo (1 n

m

), α2M-MA (50 n

m

), GST (100 n

m

), NGF-β (50 ng/ml), or vehicle (SFM) for 10 min. In the last three lanes of each panel, the indicated agent was introduced 15 min after adding GST-RAP (100 n

m

). Immunoblot analysis was performed to detect pAKT, pERK, total ERK, and β-actin. The blots shown are representative of at least three independent studies.

FIGURE 5.

FIGURE 5.

LRP-1 gene silencing in Schwann cells. Schwann cells were transfected with rat LRP-1-specific siRNA L2 (A) or with pooled NTC siRNA (B). After 48 h, cells were treated with PDGF-BB, Epo, FP6(K → A) (100 n

m

), or FP6 (100 n

m

). Protein extracts were subjected to immunoblot analysis to detect pAKT, pERK, and total ERK/MAP kinase. The blots shown are representative of four independent studies.

FIGURE 6.

FIGURE 6.

FP3 inhibits activation of Akt and ERK/MAP kinase in response to NGF-β but not FP6. PC12 cells were treated with vehicle (serum-free medium, SFM), NGF-β (50 ng/ml), FP4 (100 n

m

), FP6 (100 n

m

), FP6(K → A) (100 n

m

), or FP3 (100 n

m

) for 10 min. In the last two lanes, FP3 was preincubated with NGF-β or FP6 for 15 min at 37 °C and then added to the cell cultures. Protein extracts were subjected to immunoblot analysis to detect pAKT, pERK, and total ERK/MAP kinase. The blot shown is representative of four independent studies.

FIGURE 7.

FIGURE 7.

FP3 and FP6 have opposite effects on PC12 cell neuritogenesis. PC12 cells were cultured for 48 h in serum-free medium alone (A), or supplemented with 100 n

m

FP6 (B), 50 ng/ml NGF-β (C), 100 n

m

FP3 (D), or NGF-β+FP3 (E). Axodendritic process formation was assessed by phase contrast microscopy. Representative fields are shown (n = 3).

FIGURE 8.

FIGURE 8.

FP3 and FP6 differentially regulate expression of GAP-43. PC12 cells were cultured in serum-free medium for 5 h and then treated with vehicle (SFM), NGF-β (50 ng/ml), NGF-β + FP3 (100 n

m

), FP3 (100 n

m

), or FP6 (100 n

m

). Incubations were conducted for 4 or 8 h. Total RNA was isolated. GAP-43 mRNA expression was determined by qPCR (mean ± S.D., n = 4; *, p < 0.05, one-way analysis of variance with Tukey's posthoc for each time period).

FIGURE 9.

FIGURE 9.

FP6 and the RBD from purified α2M activate cell signaling equivalently. A, the 18-kDa RBD, isolated from α2M-MA by proteolysis and purified by chromatography, was subjected to SDS-PAGE and stained with Coomassie Blue. B, PC12 cells were cultured in SFM or treated with FP6 (50 n

m

) or the 18-kDa RBD from α2M-MA (50 n

m

) for 10 min. In some lanes, the cells were pretreated with GST-RAP (100 n

m

) for 15 min, as indicated. Protein extracts were subjected to SDS-PAGE and immunoblot analysis to detect pAKT, pERK, and total ERK/MAP kinase. The blot shown is representative of triplicate independent experiments.

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