Integrin alpha4beta1-dependent adhesion to ADAM 28 (MDC-L) requires an extended surface of the disintegrin domain.
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, alpha4beta1. To elucidate which residues comprise the alpha4beta1 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 Lys(437), Lys(442), Lys(455), Lys(459), Lys(460), Lys(469), and Glu(476) as being essential for alpha4beta1-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 alpha4beta1 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.
Bridges
Lance C
LC
Department of Biochemistry and Molecular Biology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
Hanson
Krista R
KR
Tani
Patricia H
PH
Mather
Timothy
T
Bowditch
Ron D
RD
D051722:ADAM Proteins; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D002448:Cell Adhesion; D019483:Disintegrins / Q000737:chemistry / Q000378:metabolism*; D018604:Epitope Mapping; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000276:immunology; D039041:Integrin alpha4beta1 / Q000378:metabolism*; D019169:Jurkat Cells; D008214:Lymphocytes / Q000201:enzymology; D016399:Lymphoma, T-Cell / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D019483:Disintegrins; D007141:Immunoglobulin Fc Fragments; D039041:Integrin alpha4beta1; D011994:Recombinant Proteins; D051722:ADAM Proteins; C410224:ADAM28 protein, human; C472807:Adam32 protein, mouse; D008666:Metalloendopeptidases; D000409:Alanine