Antibody-Induced Activation of 1 Integrin Receptors Stimulates cAMP-Dependent Migration of Breast Cells on Laminin-5 George E. Plopper,* ,1 Janice L. Huff,* Will L. Rust,* Martin A. Schwartz,† and Vito Quaranta‡ *Department of Biological Sciences, University of Nevada, Las Vegas, Nevada; and †Department of Vascular Biology and ‡Department of Cell Biology, The Scripps Research Institute, La Jolla, California Received January 8, 2001 The 1 integrin-stimulating antibody TS2/16 induces cAMP-dependent migration of MCF-10A breast cells on the extracellular matrix protein laminin-5. TS2/16 stimulates a rise in intracellular cAMP within 20 min after plating. Pertussis toxin, which inhibits both antibody-induced migration and cAMP accumulation, targets the Gi3 subunit of heterotrimeric G proteins in these cells, suggesting that Gi3 may link integrin activation and migration via a cAMP signaling pathway. © 2000 Academic Press Key Words: extracellular matrix; metastasis; signal transduction. Laminins are a diverse group of heterotrimeric ex- tracellular matrix proteins that constitute a major component of the basement membrane of epithelial tissues. The laminin-5 isoform, consisting of the 3, 3, and 2 subunits, is abundantly expressed in the base- ment membrane of breast tissue [1] where it plays a role in mammary branching morphogenesis, and adhe- sion and migration of breast epithelial cells [2]. Evidence from both in vitro and in vivo studies sup- port a functional role for laminin-5 in cell migration of both normal and malignant breast epithelial cells. Our laboratory has previously shown that in vitro, laminin-5 is the preferred adhesive substrate for breast epithelial cells [1]. In haptotactic migration as- says, nontumorigenic breast cell lines fail to migrate significantly on laminin-5, whereas laminin-5 supports migration of highly malignant breast cell lines. In vivo, laminin-5 expression is enhanced in invading regions of metastatic breast tumors[3]. In addition, an altered conformation of laminin-5, resulting from proteolytic cleavage of the 2 chain by matrix metalloprotease 2, is found at sites of tissue invasion, and this cleavage stimulates migration of otherwise nonmigratory breast cells in vitro [4]. Laminin-5 may contribute to the pro- gression of tumorigenic breast cells from the stationary to malignant phenotype by stimulating enhanced mi- gration of these cells. Cells interact with laminins primarily through inte- grin receptors [5]. Ligand induced signal transduction by integrin/laminin binding regulates intracellular pH, tyrosine phosphorylation, inositol lipid metabolism, and calcium (Ca 2+ ) oscillations [6]. Signaling molecules known to associate with integrins receptors include protein tyrosine kinases, serine/threonine kinases, phospholipid kinases and lipases, ion channels, and members of the rho family of small molecular weight GTP binding proteins [6]. Laminin-5 is recognized by the 31, 61, and 64 integrin receptors in a num- ber of cell types, and the functional consequence of these interactions depend on the integrin receptor en- gaged. For example, ligation of laminin-5 with the 64 integrin receptor supports branching morphogen- esis and hemidesmosome formation in breast epithelial cells [2], while interaction with 31 integrin supports migration of these same cells in vitro [7]. Little infor- mation is currently available on the specific signaling pathways triggered during these events. While investigating the role of the 31 integrin in motility of breast epithelial cells, we observed that haptotactic migration of the immortalized breast epi- thelial cell line MCF-10A on laminin-5 was stimulated by direct activation of the 1 integrin receptor with the 1-activating monoclonal antibody TS2/16. Migration was dependent on intracellular cAMP signaling, and TS2/16-promoted a rise in intracellular cAMP levels that occurred 20 min after plating on laminin-5. Mi- gration and cAMP accumulation were inhibited by treatment of the cells with pertussis toxin, a compound that inactivates the  subunit of the inhibitory class of heterotrimeric G proteins via ADP-ribosylation. We 1 To whom correspondence should be addressed at 4505 Maryland Parkway, Las Vegas, NV 89154-4004. Fax: (702) 895-3956. E-mail: plopper@nye.nscee.edu. Molecular Cell Biology Research Communications 4, 129 –135 (2000) doi:10.1006/mcbr.2001.0267, available online at http://www.idealibrary.com on 129 1522-4724/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.