title: Mutational analysis of MAdCAM-1/alpha4beta7 interactions reveals significant binding determinants in both the first and second immunuglobulin domains.
abstract: The selective emigration of blood born leukocytes into tissues is mediated, in part by interactions of Ig-like cell adhesion molecules (IgCAMs) expressed on vascular endothelium and their cognate ligands, the leukocyte integrins. Within mucosal lymphoid tissues and gastrointestinal sites the mucosal vascular addressin. MAdCAM-1 is the predominant IgCAM, mediating specific lymphocyte homing via interactions with its ligand on lymphocytes, the integrin alpha4beta7. Previous studies have shown that an essential binding motif resides in the first Ig domain of all IgCAMs, containing an acidic residue (D or E) preceded by an aliphatic residue (L or I) that resides in strand C or the CD loop. However, domain swap experiments with MAdCAM-1 and VCAM-1 have shown a requirement for both Ig domains 1 and 2 for efficient integrin binding. We describe the use of chimeric MAdCAM-1/VCAM-1 receptors and point mutations in MAdCAM-1 to define other sites that are required for binding to the integrin alpha4beta7. We find that, in addition to critical CD loop residues, other regions in both domain one and two contribute to MAdCAM-1/alpha4beta7 interactions, including a buried arginine residue in the F strand of domain one and several acidic residues in a highly extended DE ribbon in domain 2. These mutations, when placed in the recently solved crystal structure of human MAdCAM-1 give insight into the integrin binding preference of this unique receptor.
authors.affiliation: LeukoSite Inc, Cambridge, MA 02142, USA.
authors.forename: N
authors.initials: N
authors.lastname: Green
authors.forename: J
authors.initials: J
authors.lastname: Rosebrook
authors.forename: N
authors.initials: N
authors.lastname: Cochran
authors.forename: K
authors.initials: K
authors.lastname: Tan
authors.forename: J H
authors.initials: JH
authors.lastname: Wang
authors.forename: T A
authors.initials: TA
authors.lastname: Springer
authors.forename: M J
authors.initials: MJ
authors.lastname: Briskin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion / Q000235:genetics; D015815:Cell Adhesion Molecules; D006224:Cricetinae; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016023:Integrins / Q000737:chemistry / Q000378:metabolism*; D007962:Leukocytes / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009088:Mucoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D016201:Receptors, Lymphocyte Homing / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D019010:Vascular Cell Adhesion Molecule-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D015815:Cell Adhesion Molecules; D007136:Immunoglobulins; D016023:Integrins; C504265:MADCAM1 protein, human; D009088:Mucoproteins; D016201:Receptors, Lymphocyte Homing; D011993:Recombinant Fusion Proteins; D019010:Vascular Cell Adhesion Molecule-1; C071777:integrin alpha4beta7