A Novel Monoclonal Antibody Recognizing a Cation-Dependent Epitope within the Regulatory Loop of Human β 1 Integrin (CD29)

Abstract

Cell adhesion receptors of the integrin superfamily can be expressed in different affinity states towards their ligands. It has been previously demonstrated that β₁ integrins α4β₁ and α5β₁ 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-β₁ integrin monoclonal antibodies (MAbs). In both instances, the activation of β₁ integrins is believed to be the result of conformational changes propagating along the β₁ integrin chain which in turn increase accessibility to the ligand. A cluster of either function-activating or function-inhibiting anti-β₁ integrin MAbs have been shown to bind within a 12 amino acid long regulatory loop between residues 207 and 218 of the human β₁ 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 Ca²⁺ or Mn²⁺ but not Mg²⁺. In addition, the activation of α4β₁ and α5β₁ integrins by 96.9H9 is a two-step process with distinct cation requirements. Whereas Ca²⁺ is sufficient to promote binding of the antibody to the β₁ integrin chain, Mg²⁺ is necessary for activating function following 96.9H9 binding. Our data therefore suggest that the regulatory epitope of the human β₁ integrin chain is flexible with multiple conformations according to the cationic environment.