Binding of Purified Collagen Receptors (α1β1, α2β1) and RGD-Dependent Integrins to Laminins and Laminin Fragments

Integrins α1β1 and α2β1, when purified by collagen affinity chromatography, showed distinct binding to mouse tumor laminin-1, which has the chain composition α1β1γ1. The binding was, however, about 10-fold lower than to collagen IV. Only little (α1β1) or no binding (α2β1) was observed to two different laminin isoforms (α2β1γ1, α2β2γ1) from human placenta. Binding to laminin-1 was abolished by EDTA and could be specifically inhibited by antibodies to the respective integrin a subunit. These antibodies also inhibited cell adhesion to collagens. The binding of soluble integrins was weaker than that of immobilized integrins but could be enhanced by an activating anti(β1 integrin). No enhancement was observed for immobilized integrins. Studies with laminin-1 fragments demonstrated lack of binding to the major cell-adhesive fragment E8 from the long arm, fragments E3 and E4, involved in heparin-binding and self-assembly, respectively, and fragment P1, corresponding to the inner segments of the short arms. A larger short-arm fragment (E1XNd), which lacks the N-terminal β1 chain domains V and VI, was as active as laminin. Together, these results, suggested the localization of the binding sites for α1β1 and α2β1 to the N-terminal region of the laminin α1 chain. Fragment P1 but not intact laminin-1 bound to αVβ3 integrin in an EDTA-sensitive and RGD-sensitive manner, underscoring previous data on the cryptic nature of the RGD site in laminin-1. Further analyses by surface plasmon resonance assays demonstrated a KD= 50 nM for α2β1/laminin-1 binding and a KD= 450 nM for αVβ3/fragment P1 binding and confirmed the anti-β1-mediated increase in affinity for α2β1.