Localization of Cranin (Dystroglycan) at Sites of Cell-Matrix and Cell-Cell Contact: Recruitment to Focal Adhesions Is Dependent upon Extracellular Ligands

Abstract

We report that cranin (dystroglycan) can become recruited to focal adhesions of cultured rat REF 52 fibroblasts and human aortic smooth muscle cells. Within mature focal adhesions, cranin was present within the plaque region defined by β1 integrin, vinculin and phosphotyrosine staining, but occupied a larger domain corresponding to, the terminal segments of stress fibers that was more precisely co-extensive with the cytoskeletal proteins alpha-actinin, utrophin and aciculin. When REF 52 fibroblasts were plated on different substrata in the absence of protein synthesis and secretion in serum-free medium, focal clusters of cranin readily formed within 2 hours on matrix proteins that bind cranin directly (laminin or agrin) which were maintained as the focal adhesions became mature. In contrast, cranin failed to become targeted to cell-substratum attachment sites, either at early or later times. when cells were plated on a variety of other substrata that elicit formation of focal adhesions but do not bind cranin directly (fibronectin, vitronectin, collagen type IV, or anti-β integrin antibody TS2/16). These data strongly suggest that targeting of cranin to focal adhesions was dependent upon the presence of an extracellular ligand capable of binding cranin directly. How-ever, some cultured nonmuscle cell lines (e.g., human umbilical vein endothelial cells, NIH 3T3 and CHO cells) failed to localize cranin to focal adhesions, even when plated on laminin. Cranin was also enriched at cell-cell adherens-type junctions of human normal breast MCF-10 epithelial cells, and at growth cones of E17 rat hippocampal axons. That cranin can become targeted to sites of cell-cell and cell-substratum contact in diverse cell types supports the hypothesis that cranin may be involved in mediating or regulating cell adhesion. The absence of muscle-specific and synapse-specific proteins within fibroblasts and epithelial cells provides a different context for thinking about cranin (dystroglycan) that may aid in discerning general principles of its structure and function.

Key Words:

• Cell Adhesion
• Extracellular Matrix
• Laminin
• Agrin
• Utrophin
• Aciculin