title: Probing the interaction of tetraspanin CD151 with integrin alpha 3 beta 1 using a panel of monoclonal antibodies with distinct reactivities toward the CD151-integrin alpha 3 beta 1 complex.
abstract: CD151, a member of the tetraspanin family of proteins, forms a stable complex with integrin alpha 3 beta 1 and regulates integrin-mediated cell-substrate adhesion. However, the molecular basis of the stable association of CD151 with integrin alpha 3 beta 1 remains poorly understood. In the present study, we show that a panel of anti-human CD151 mAbs (monoclonal antibodies) could be divided into three groups on the basis of their abilities to co-immunoprecipitate integrin alpha 3: Group-1 mAbs were devoid of sufficient activities to co-precipitate integrin alpha 3 under both low- and high-stringency detergent conditions; Group-2 mAbs co-precipitated integrin alpha 3 under low-stringency conditions; and Group-3 mAbs exhibited strong co-precipitating activities under both conditions. Group-1 mAbs in particular exhibited increased reactivity toward integrin alpha 3 beta 1-unbound CD151, indicating that the binding sites for Group-1 mAbs are partly blocked by bound integrin alpha 3 beta 1. Epitope mapping using a series of CD151 mutants with substitutions at amino acid residues that are not conserved between human and mouse CD151 revealed that Gly(176)/Gly(177), Leu(191) and Gln(194) comprise epitopes characteristic of Group-1 mAbs. Replacement of short peptide segments, each containing one of these epitopes, with those of other tetraspanins lacking stable interactions with integrin alpha 3 beta 1 demonstrated that the segment from Cys(185) to Cys(192), including Leu(191), was involved in the stable association of CD151 with integrin alpha 3 beta 1, as was the Gln(194)-containing QRD peptide. Taken together these results indicate that two consecutive segments including two Group-1 epitopes, Leu(191) and Gln(194), comprise an interface between CD151 and integrin alpha 3 beta 1, and, along with the epitope including Gly(176)/Gly(177), are concealed by bound integrin.
authors.affiliation: Laboratory of Extracellular Matrix Biochemistry, Division of Protein Chemistry, Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Yamada
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Tamura
authors.forename: Noriko
authors.initials: N
authors.lastname: Sanzen
authors.forename: Ryoko
authors.initials: R
authors.lastname: Sato-Nishiuchi
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Hasegawa
authors.forename: Leonie K
authors.initials: LK
authors.lastname: Ashman
authors.forename: Eric
authors.initials: E
authors.lastname: Rubinstein
authors.forename: María
authors.initials: M
authors.lastname: Yáñez-Mó
authors.forename: Francisco
authors.initials: F
authors.lastname: Sánchez-Madrid
authors.forename: Kiyotoshi
authors.initials: K
authors.lastname: Sekiguchi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015703:Antigens, CD / Q000276:immunology* / Q000378:metabolism; D001665:Binding Sites; D045744:Cell Line, Tumor; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D006801:Humans; D039201:Integrin alpha3beta1 / Q000276:immunology*; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D060185:Tetraspanin 24
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C554715:CD151 protein, human; C554716:Cd151 protein, mouse; D000939:Epitopes; D039201:Integrin alpha3beta1; D060185:Tetraspanin 24