title: Analysis of 4-1BBL and laminin binding to murine 4-1BB, a member of the tumor necrosis factor receptor superfamily, and comparison with human 4-1BB.
abstract: The T cell activation antigen 4-1BB (CDw137) is a distantly related member of the tumor necrosis factor receptor family of cell surface receptors. We previously reported that murine 4-1BB (m4-1BB) bound to extracellular matrix (ECM) proteins. Recently, a tumor necrosis factor-like ligand of m4-1BB, m4-1BBL, as well as the human counterparts of 4-1BB (ILA) and 4-1BBL (h4-1BB and h4-1BBL, respectively) have been cloned. No information is currently available on how binding of m4-1BB to ECM proteins affects its binding to m4-1BBL and vice versa and if the ability of m4-1BB to bind ECM proteins is conserved across species. We report that binding of m4-1BBL to m4-1BB blocked its ability to bind laminin (LN), while binding of m4-1BB to LN did not block its ability to bind m4-1BBL. Furthermore, binding of m4-1BBL to the m4-1BB.LN complex did not displace LN. These findings suggest the two ligands bind to proximal but distinct sites on m4-1BB. This is supported by the observation that six of eight anti-m4-1BB monoclonal antibodies blocked the interaction between 4-1BB and 4-1BBL, while seven blocked LN binding. Ligand and monoclonal antibody binding studies with a truncated protein lacking the amino-terminal LN-homologous domain of m4-1BB demonstrated that regions downstream of the LN-homologous domain participate in LN binding and that the intact protein is required for m4-1BBL binding. Studies with h4-1BB showed that h4-1BB only bound h4-1BBL, indicating that the ECM binding activity of 4-1BB is not conserved across species. This finding allowed the construction of murine/human 4-1BB chimeras, which permitted further dissection of the regions of 4-1BB involved in LN and 4-1BBL binding and suggests that sequence differences in the LN-homologous domain of h4-1BB in part account for the inability of h4-1BB to bind ECM proteins.
authors.affiliation: Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121, USA.
authors.forename: D T
authors.initials: DT
authors.lastname: Loo
authors.forename: N J
authors.initials: NJ
authors.lastname: Chalupny
authors.forename: J
authors.initials: J
authors.lastname: Bajorath
authors.forename: W W
authors.initials: WW
authors.lastname: Shuford
authors.forename: R S
authors.initials: RS
authors.lastname: Mittler
authors.forename: A
authors.initials: A
authors.lastname: Aruffo
mesh_terms: D053330:4-1BB Ligand; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D019556:COS Cells; D005110:Extracellular Space; D006801:Humans; D007797:Laminin / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D053330:4-1BB Ligand; D015703:Antigens, CD; D007797:Laminin; D008024:Ligands; D017475:Receptors, Nerve Growth Factor; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; C505714:TNFRSF9 protein, human; C505974:TNFSF9 protein, human; C505715:Tnfrsf9 protein, mouse; C505975:Tnfsf9 protein, mouse; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9; D014409:Tumor Necrosis Factor-alpha