A novel monoclonal antibody recognizing a cation-dependent epitope within the regulatory loop of human beta(1) integrin (CD29).
Cell adhesion receptors of the integrin superfamily can be expressed in different affinity states towards their ligands. It has been previously demonstrated that beta(1) integrins alpha4beta(1) and alpha5beta(1) are expressed in a nonligand binding form by human hemopoietic progenitor cells but can be activated into a ligand binding form by a variety of stimuli including intracellular stimuli generated by cytokine receptors and extracellular stimuli generated by function-activating anti-beta(1) integrin monoclonal antibodies (MAbs). In both instances, the activation of beta(1) integrins is believed to be the result of conformational changes propagating along the beta(1) integrin chain which in turn increase accessibility to the ligand. A cluster of either function-activating or function-inhibiting anti-beta(1) integrin MAbs have been shown to bind within a 12 amino acid long regulatory loop between residues 207 and 218 of the human beta(1) integrin chain. We describe in this report the first MAb (96.9H9) specific for this regulatory loop whose binding is cation-dependent and requires either Ca(2+) or Mn(2+) but not Mg(2+). In addition, the activation of alpha4beta(1) and alpha5beta(1) integrins by 96.9H9 is a two-step process with distinct cation requirements. Whereas Ca(2+) is sufficient to promote binding of the antibody to the beta(1) integrin chain, Mg(2+) is necessary for activating function following 96.9H9 binding. Our data therefore suggest that the regulatory epitope of the human beta(1) integrin chain is flexible with multiple conformations according to the cationic environment.
Lévesque
Jean-Pierre
JP
The Peter MacCallum Cancer Institute, Stem Cell Biology Laboratory, East Melbourne, Australia. jp.levesque@pmci.unimelb.edu.au
Takada
Yoshikazu
Y
Puzon-McLaughlin
Wilma
W
Simmons
Paul J
PJ
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001665:Binding Sites; D016466:CHO Cells; D002413:Cations, Divalent / Q000378:metabolism; D002448:Cell Adhesion / Q000276:immunology; D002460:Cell Line; D006224:Cricetinae; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D019012:Integrin beta1 / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D008024:Ligands; D051379:Mice; D008807:Mice, Inbred BALB C; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; D000939:Epitopes; D005353:Fibronectins; D019012:Integrin beta1; D008024:Ligands; D011993:Recombinant Fusion Proteins