A specific alpha5beta1-integrin conformation promotes directional integrin translocation and fibronectin matrix formation.
ABSTRACT Integrin adhesion receptors are structurally dynamic proteins that adopt a number of functionally relevant conformations. We have produced a conformation-dependent anti-alpha5 monoclonal antibody (SNAKA51) that converts alpha5beta1 integrin into a ligand-competent form and promotes fibronectin binding. In adherent fibroblasts, SNAKA51 preferentially bound to integrins in fibrillar adhesions. Clustering of integrins expressing this activation epitope induced directional translocation of alpha5beta1, mimicking fibrillar adhesion formation. Priming of alpha5beta1 integrin by SNAKA51 increased the accumulation of detergent-resistant fibronectin in the extracellular matrix, thus identifying an integrin conformation that promotes matrix assembly. The SNAKA51 epitope was mapped to the calf-1/calf-2 domains. We propose that the action of the antibody causes the legs of the integrin to change conformation and thereby primes the integrin to bind ligand. These findings identify SNAKA51 as the first anti-integrin antibody to selectively recognize a subset of adhesion contacts, and they identify an integrin conformation associated with integrin translocation and fibronectin matrix formation.
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ABSTRACT: Integrins are alphabeta heterodimeric receptors that mediate divalent cation-dependent cell-cell and cell-matrix adhesion through tightly regulated interactions with ligands. We have solved the crystal structure of the extracellular portion of integrin alphaVbeta3 at 3.1 A resolution. Its 12 domains assemble into an ovoid "head" and two "tails." In the crystal, alphaVbeta3 is severely bent at a defined region in its tails, reflecting an unusual flexibility that may be linked to integrin regulation. The main inter-subunit interface lies within the head, between a seven-bladed beta-propeller from alphaV and an A domain from beta3, and bears a striking resemblance to the Galpha/Gbeta interface in G proteins. A metal ion-dependent adhesion site (MIDAS) in the betaA domain is positioned to participate in a ligand-binding interface formed of loops from the propeller and betaA domains. MIDAS lies adjacent to a calcium-binding site with a potential regulatory function.Science 11/2001; 294(5541):339-45. · 31.20 Impact Factor
Article: Integrin structure.[show abstract] [hide abstract]
ABSTRACT: The integrins are a family of alpha,beta heterodimeric receptors that mediate dynamic linkages between extracellular adhesion molecules and the intracellular actin cytoskeleton. Integrins are expressed by all multicellular animals, but their diversity varies widely among species; for example, in mammals, 19 alpha and 8 beta subunit genes encode polypeptides that combine to form 25 different receptors, whereas the Drosophila and Caenorhabditis genomes encode only five and two integrin alpha subunits respectively. Thousands of studies over the last two decades have investigated the molecular, cellular and organismal basis of integrin function. Gene deletion has demonstrated essential roles for almost all integrins, with the defects suggesting widespread contributions to both the maintenance of tissue integrity and the promotion of cellular migration. Integrin-ligand interactions are now considered to provide physical support for cells in order to maintain cohesion, to permit the generation of traction forces to enable movement, and to organize signalling complexes to modulate differentiation and cell fate. Animal-model studies have also shown that integrins contribute to the progression of many common diseases, and have implicated them as potential therapeutic targets. The use of anti-integrin monoclonal antibodies and ligand-mimetic peptides has validated this suggestion for inflammatory, neoplastic, traumatic and infectious conditions. Thus, to understand more about the mechanisms underlying tissue organization and cellular trafficking, and to identify approaches for regulating these processes in disease, there is intense interest in determining the molecular basis of integrin function. It is important to state at the outset that the tertiary structure of the integrin dimer is unknown. Our current understanding of the molecular basis of integrin function is therefore compiled from the results of a large number of studies that have employed a wide range of complementary technologies.Biochemical Society Transactions 02/2000; 28(4):311-39. · 3.71 Impact Factor
Article: Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization.[show abstract] [hide abstract]
ABSTRACT: We have developed two rat mAbs that recognize different subunits of the human fibroblast fibronectin receptor complex and have used them to probe the function of this cell surface heterodimer. mAb 13 recognizes the integrin class 1 beta polypeptide and mAb 16 recognizes the fibronectin receptor alpha polypeptide. We tested these mAbs for their inhibitory activities in cell adhesion, spreading, migration, and matrix assembly assays using WI38 human lung fibroblasts. mAb 13 inhibited the initial attachment as well as the spreading of WI38 cells on fibronectin and laminin substrates but not on vitronectin. Laminin-mediated adhesion was particularly sensitive to mAb 13. In contrast, mAb 16 inhibited initial cell attachment to fibronectin substrates but had no effect on attachment to either laminin or vitronectin substrates. When coated on plastic, both mAbs promoted WI38 cell spreading. However, mAb 13 (but not mAb 16) inhibited the radial outgrowth of cells from an explant on fibronectin substrates. mAb 16 also did not inhibit the motility of individual fibroblasts on fibronectin in low density culture and, in fact, substantially accelerated migration rates. In assays of the assembly of an extracellular fibronectin matrix by WI38 fibroblasts, both mAbs produced substantial inhibition in a concentration-dependent manner. The inhibition of matrix assembly resulted from impaired retention of fibronectin on the cell surface. Treatment of cells with mAb 16 also resulted in a striking redistribution of cell surface fibronectin receptors from a streak-like pattern to a relatively diffuse distribution. Concomitant morphological changes included decreases in thick microfilament bundle formation and reduced adhesive contacts of the streak-like and focal contact type. Our results indicate that the fibroblast fibronectin receptor (a) functions in initial fibroblast attachment and in certain types of adhesive contact, but not in the later steps of cell spreading; (b) is not required for fibroblast motility but instead retards migration; and (c) is critically involved in fibronectin retention and matrix assembly. These findings suggest a central role for the fibronectin receptor in regulating cell adhesion and migration.The Journal of Cell Biology 09/1989; 109(2):863-75. · 10.26 Impact Factor