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J. Biol. Chem., Vol. 283, Issue 29, 19904-19911, July 18, 2008

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Molecular Dissection of the Human ₂-Macroglobulin Subunit Reveals Domains with Antagonistic Activities in Cell Signaling^*

From the Departments of Pathology and Anesthesiology, University of California, San Diego, La Jolla, California 92093

₂-Macroglobulin (₂M) 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 ₂M are responsible for its various functions, we hypothesized that the overall effects of ₂M 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 Lys¹³⁷⁰ and Lys¹³⁷⁴, which precludes LRP-1 binding. FP3 blocked activation of Akt and ERK/MAP kinase in response to nerve growth factor-β (NGF-β) 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-β 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 ₂M by proteolysis and chromatography. We propose that the effects of intact ₂M 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.

Received for publication, March 5, 2008 , and in revised form, May 8, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants R01 NS-054671, R01 HL-60551, and R01 NS-057456. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: UCSD School of Medicine, Dept. of Pathology, 9500 Gilman Dr., La Jolla, CA 92093-0612. Tel.: 858-534-1887; Fax: 858-534-0414; E-mail: sgonias{at}ucsd.edu.

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