BINDING OF PURIFIED COLLAGEN RECEPTORS (ALPHA-1-BETA-1, ALPHA-2-BETA-1) AND RGD-DEPENDENT INTEGRINS TO LAMININS AND LAMININ FRAGMENTS

Integrins alpha 1 beta 1 and alpha 2 beta 1, when purified by collagen affinity chromatography, showed distinct binding to mouse tumor lamin in-1, which has the chain composition alpha 1 beta 1 gamma. The bindin g was, however, about 10-fold lower than to collagen IV. Only little ( alpha 1 beta 1) or no binding (alpha 2 beta 1) was observed to two dif ferent laminin isoforms (alpha 2 beta 1 gamma 1, alpha 2 beta 2 gamma 1) from human placenta. Binding to laminin-1 was abolished by EDTA and could be specifically inhibited by antibodies to the respective integ rin ex subunit. These antibodies also inhibited cell adhesion to colla gens. The binding of soluble integrins was weaker than that of immobil ized integrins but could be enhanced by an activating anti(beta 1 inte grin>. No enhancement was observed for immobilized integrins. Studies with laminin-1 fragments demonstrated lack of binding to the major cel l-adhesive fragment E8 from the long arm, fragments E3 and E4, involve d in heparin-binding and self-assembly, respectively, and fragment P1, corresponding to the inner segments of the shea arms. A larger short arm fragment (E1XNd), which lacks the N-terminal beta 1 chain domains V and VI, was as active as laminin. Together, these results, suggested the localization of the binding sites for alpha 1 beta 1 and alpha 2 beta 1 to the N-terminal region of the laminin alpha 1 chain. Fragment P1 but not intact laminin-1 bound to alpha V beta 3 integrin in an ED TA-sensitive and RGD-sensitive manner, underscoring previous data on t he cryptic nature of the RGD site in laminin-1. Further analyses by su rface plasmon resonance assays demonstrated a K-D = 50 nM for alpha 2 beta 1/laminin-1 binding and a K-D = 450 nM for alpha V beta 3/fragmen t P1 binding and confirmed the anti-beta 1-mediated increase in affini ty for alpha 2 beta 1.