Entactin is an extracellular matrix glycoprotein which binds to laminin and is found in most renal basement membranes and in the glomerular mesangial matrix. In the present study, we have characterized specific integrin receptors on cultured human mesangial cells (CHMC) responsible for adhesion to native entactin. The integrin receptors α2β1, α3β1, α5β1, αvβ3, αvβ5, and α6 complexed with either β1 or β4 could be immune precipitated from detergent extracts of metabolically labeled CHMC. Adhesion assays with inhibitory anti integrin monoclonal antibodies (mab) demonstrated that CHMC use both αvβ3 and a β1-containing integrin to bind surfaces coated with native entactin. Optimal binding of CHMC to native entactin required the participation of cations. Using wild type and mutant recombinant entactin fragments, the binding site for the αvβ3 receptor was localized to the RGD sequence on the rod or E domain of entactin. CHMC adhesion to mutant full length recombinant entactin ligands lacking the E domain RGD sequence confirmed the presence of ligand binding site(s) for β1 integrin receptor(s). Differences in CHMC binding characteristics to recombinant and full length entactin compared to native bovine basement membrane entactin were observed. This suggests that tertiary molecular structure may contribute to entactin ligand binding properties. Primary amino acid residue sequences and tertiary structure of entactin may play roles in forming functional cell attachment sites in native basement membrane entactin.
Bibliographical noteFunding Information:
Patricia Erickson in preparation of this manuscript. This work was supported by grants from the Juvenile Diabetes Foundation International, American Diabetes Foundation, Minnesota Affiliate (Dr. Alfred Fish), National Institutes of Health DC00133, (Dr. Youngki Kim), National Institutes of Health DK13083 (Dr. S. Michael Mauer), National Institutes of Health A1 10704 (Alfred F. Michael), American Cancer Society Grant #1M-687 (Dr. Elizabeth A. Wayner) and the Vikings' Children Fund. Dr. Jin received a research fellowship from the Samsung Medical Center, Seoul, Korea.
- Mesangial cells