We will characterize the ligand specificity, ligand binding sites, regulation of affinity for ligand, and cytoskeletal association of the leukocyte adhesion receptors Mac-l and p150,95. Mac-l is both a complement receptor (CR3) and a cell adhesion receptor; p150,95 is a closely related adhesion receptor expressed on myeloid cells and abundantly on hairy leukemia cells. Genetic deficiency of these receptors results in recurring bacterial infections which are often fatal in childhood. The alpha subunits of these receptors control ligand specificity, and we hypothesize that tandem repeats which contain putative divalent cation binding sites are the ligand binding sites. To define ligand binding sites, we will cut and paste alpha subunit cDNA clones to prepare hybrid Mac-l x LFA-l and p150,95 x LFA-l alpha subunits which will be expressed using retroviral vectors in association with the common beta subunit on the surface of T lymphoma cells. MAb epitopes will be localized and correlated with MAb effect on function. Binding to iC3b in solution or on E; and binding to novel cellular ligands in solution, artificial planar membranes, and on intact cells will define the binding sites specific for different ligands, and the contribution of these sites to affinity. Similarly expressed truncated alpha subunits will be used to define the importance of transmembrane and cytoplasmic domains in regulating affinity for ligand and cytoskeletal association. We hypothesize that cells dynamically regulate adhesion receptor affinity for ligand. This will be tested for native molecules on neutrophils and for recombinant hybrid and truncated molecules expressed on T lymphoma cells by Scatchard binding of monomeric iC3b and cell surface ligands, after stimulation with fMLP or phorbol esters. Cytoskeletal association will be measured in parallel by detergent solubilization. In parallel with the above studies, novel ligand molecules recognized by Mac-l in neutrophil aggregation and adhesion to endothelial cells will be defined with MAb. Ligand molecules will be characterized and primary sequence defined at the protein and cDNA levels. Recognition sequences, hypothesized to be RGD-like, will be defined. Purified natural and recombinant secreted molecules will be used in the shove ligand binding studies. The intracellular storage site of fMLP-regulatable Mac- l in neutrophils and s murine Mae-l cDNA clone will also be characterized.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37CA031799-11
Application #
3482229
Study Section
Immunobiology Study Section (IMB)
Project Start
1988-05-01
Project End
1993-04-30
Budget Start
1991-05-01
Budget End
1992-04-30
Support Year
11
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Immune Disease Institute, Inc.
Department
Type
DUNS #
115524410
City
Boston
State
MA
Country
United States
Zip Code
02115
Luo, Bing-Hao; Carman, Christopher V; Springer, Timothy A (2007) Structural basis of integrin regulation and signaling. Annu Rev Immunol 25:619-47
Springer, Timothy A (2006) Complement and the multifaceted functions of VWA and integrin I domains. Structure 14:1611-6
Vorup-Jensen, Thomas; Carman, Christopher V; Shimaoka, Motomu et al. (2005) Exposure of acidic residues as a danger signal for recognition of fibrinogen and other macromolecules by integrin alphaXbeta2. Proc Natl Acad Sci U S A 102:1614-9
Vorup-Jensen, Thomas; Ostermeier, Christian; Shimaoka, Motomu et al. (2003) Structure and allosteric regulation of the alpha X beta 2 integrin I domain. Proc Natl Acad Sci U S A 100:1873-8
Springer, Timothy A (2002) Predicted and experimental structures of integrins and beta-propellers. Curr Opin Struct Biol 12:802-13
Takagi, Junichi; Springer, Timothy A (2002) Integrin activation and structural rearrangement. Immunol Rev 186:141-63
Shimaoka, Motomu; Takagi, Junichi; Springer, Timothy A (2002) Conformational regulation of integrin structure and function. Annu Rev Biophys Biomol Struct 31:485-516
Shimaoka, Motomu; Lu, Chafen; Salas, Azucena et al. (2002) Stabilizing the integrin alpha M inserted domain in alternative conformations with a range of engineered disulfide bonds. Proc Natl Acad Sci U S A 99:16737-41
Zang, Q; Springer, T A (2001) Amino acid residues in the PSI domain and cysteine-rich repeats of the integrin beta2 subunit that restrain activation of the integrin alpha(X)beta(2). J Biol Chem 276:6922-9
Lu, C; Takagi, J; Springer, T A (2001) Association of the membrane proximal regions of the alpha and beta subunit cytoplasmic domains constrains an integrin in the inactive state. J Biol Chem 276:14642-8

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