title: The role of the CPNKEKEC sequence in the beta(2) subunit I domain in regulation of integrin alpha(L)beta(2) (LFA-1).
abstract: The alpha(L) I (inserted or interactive) domain of integrin alpha(L)beta(2) undergoes conformational changes upon activation. Recent studies show that the isolated, activated alpha(L) I domain is sufficient for strong ligand binding, suggesting the beta(2) subunit to be only indirectly involved. It has been unclear whether the activity of the alpha(L) I domain is regulated by the beta(2) subunit. In this study, we demonstrate that swapping the disulfide-linked CPNKEKEC sequence (residues 169-176) in the beta(2) I domain with a corresponding beta(3) sequence, or mutating Lys(174) to Thr, constitutively activates alpha(L)beta(2) binding to ICAM-1. These mutants do not require Mn(2+) for ICAM-1 binding and are insensitive to the inhibitory effect of Ca(2+). We have also localized a component of the mAb 24 epitope (a reporter of beta(2) integrin activation) in the CPNKEKEC sequence. Glu(173) and Glu(175) of the beta(2) I domain are identified as critical for mAb 24 binding. Because the epitope is highly expressed upon beta(2) integrin activation, it is likely that the CPNKEKEC sequence is exposed or undergoes conformational changes upon activation. Deletion of the alpha(L) I domain did not eliminate the mAb 24 epitope. This confirms that the alpha(L) I domain is not critical for mAb 24 binding, and indicates that mAb 24 detects a change expressed in part in the beta(2) subunit I domain. These results suggest that the CPNKEKEC sequence of the beta(2) I domain is involved in regulating the alpha(L) I domain.
authors.affiliation: Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. kamata@sc.itc.keio.ac.jp
authors.forename: Tetsuji
authors.initials: T
authors.lastname: Kamata
authors.forename: Kenneth Khiem
authors.initials: KK
authors.lastname: Tieu
authors.forename: Takehiko
authors.initials: T
authors.lastname: Tarui
authors.forename: Wilma
authors.initials: W
authors.lastname: Puzon-McLaughlin
authors.forename: Nancy
authors.initials: N
authors.lastname: Hogg
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Takada
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D018821:CD18 Antigens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016466:CHO Cells; D002448:Cell Adhesion; D017124:Conserved Sequence; D006224:Cricetinae; D004220:Disulfides / Q000737:chemistry; D000939:Epitopes / Q000276:immunology; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D016169:Lymphocyte Function-Associated Antigen-1 / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D000911:Antibodies, Monoclonal; D018821:CD18 Antigens; D004220:Disulfides; D000939:Epitopes; D016169:Lymphocyte Function-Associated Antigen-1; D018799:Intercellular Adhesion Molecule-1