Integrin α4β1-Dependent Adhesion to ADAM 28 (MDC-L) Requires an Extended Surface of the Disintegrin Domain

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Abstract
ADAMs (a disintegrin and metalloprotease) are a family of proteins that possess functional adhesive and proteolytic domains. ADAM 28 (MDC-L) is expressed by human lymphocytes and contains a disintegrin-like domain that serves as a ligand for the leukocyte integrin, α4β1. To elucidate which residues comprise the α4β1 binding site in the ADAM 28 disintegrin domain, a charge-to-alanine mutagenesis strategy was utilized. Each alanine substitution mutant was evaluated and compared to the native sequence for its ability to support cell adhesion of the T-lymphoma cell line, Jurkat. This approach identified ADAM 28 residues Lys437, Lys442, Lys455, Lys459, Lys460, Lys469, and Glu476 as being essential for α4β1-dependent cell adhesion. The epitope for a function-blocking monoclonal antibody, Dis 1-1, was localized to the N-terminal end of the ADAM 28 disintegrin domain using these same charge-to-alanine mutants. Three distinct molecular models based upon the known structures of snake venom disintegrins suggested that residues contributing to α4β1 recognition are aligned on one face of the domain. This study demonstrates that residues located outside of the disintegrin loop participate in integrin recognition of mammalian disintegrins.