Mapping of network-forming, heparin-binding, and alpha 1 beta 1 integrin-recognition sites within the alpha-chain short arm of laminin-1.
Cell-interactive and architecture-forming functions are associated with the short arms of basement membrane laminin-1. To map and characterize these functions, we expressed recombinant mouse laminin-1 alpha-chain extending from the N terminus through one third of domain IIIb. This dumbbell-shaped glycoprotein (r alpha 1(VI-IVb)'), secreted by mammalian cells, was found to possess three activities. 1) Laminin polymerization was quantitatively inhibited by recombinant protein, supporting an alpha-chain role for a three-short arm interaction model of laminin self-assembly. 2) r alpha 1(VI-IVb)' bound to heparin, and the activity was localized to a subfragment corresponding to domain VI by 125I-heparin blotting. 3) PC12 rat pheochromocytoma cells adhered to, and rapidly extended branching neurites on, r alpha 1(VI-IVb)', with adhesion inhibited by alpha 1 and beta 1 integrin chain-specific antibodies. The ability of anti-laminin antibody to block PC12 cell adhesion to laminin was selectively prevented by absorption with r alpha 1(VI-IVb)' or alpha-chain domain VI fragment. This active integrin-recognition site could furthermore be distinguished from a second cryptic alpha 1 beta 1-binding site exposed by heat treatment of fragment P1', a short arm fragment lacking globules. Thus, a polymer-forming, a heparin-binding, and the active alpha 1 beta 1 integrin-recognition site are all clustered at the end of the alpha-chain short arm, the latter two resident solely in domain VI.
Colognato-Pyke
H
H
Department of Pathology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
O'Rear
J J
JJ
Yamada
Y
Y
Carbonetto
S
S
Cheng
Y S
YS
Yurchenco
P D
PD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D039222:Integrin alpha1beta1; D016023:Integrins / Q000378:metabolism*; D007797:Laminin / Q000737:chemistry* / Q000502:physiology*; D008969:Molecular Sequence Data; D016501:Neurites / Q000502:physiology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry
D039222:Integrin alpha1beta1; D016023:Integrins; D007797:Laminin; D011994:Recombinant Proteins; D006493:Heparin