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LEUKOCYTE ADHESION RECEPTORS MAC 1 AND P150,95


Biography

Overview
The leukocyte integrins alphaMbeta32 and alphaXbeta2 are widely important in inflammatory responses of myeloid cells. They bind ligands including ICAM-1, iC3b and fibrin. Upon cell activation, integrins change shape and can bind ligand, a phenomenon known as inside-out signaling. Understanding the molecular basis of this structural alteration is of great importance. The N-terminal domains of integrin alpha and beta subunits fold together into the globular, ligand-binding headpiece. The C-terminal domains of alpha and beta form two stalks that connect to the membrane and link signals in the cytoplasm to structural alterations in the ligand-binding domains, alphaM and alphaX contain a predicted beta-propeller domain and an inserted or I domain. The I domain binds ligand through a metal ion-dependent adhesion site (MIDAS). A conformational shift between open and closed conformers is linked to a 10 A movement of the C-terminal a-helix of the I domain and regulates ligand binding at the MIDAS. The betab2 subunit contains an I-like domain with a predicted structure similar to the I domain, which also functions in ligand binding. We can lock I domains in the open or closed conformation by mutations in the hydrophobic core, or by introduction of a disulfide bond. Integrins with locked open I domains will be used to test the hypothesis that the beta-subunit 1-like domain regulates ligand binding indirectly, by altering the conformation of other domains that bind ligands directly. We will test whether the open conformation of the I domain is linked to opening of the stalk region by examining binding of reporter antibodies to the cysteine-rich repeats of the b2 subunit stalk region. We will examine if mutational activation or inactivation of integrins are associated with changes in clustering in the membrane. To test the hypothesis that in a/b heterodimers, a 10A downward movement in the C-terminal a-helix of the a-subunit I domain or 13-subunit I-like domain activates ligand binding. We will add or subtract turns of a-helix in their C-terminal a-helices, and examine effects on ligand binding and opening of the stalk region. To examine whether conformational change activates ligand binding, we will measure the kinetics and affinity of wild type and open mutant I domains with ligands including ICAM-I and iC3b, and test the hypothesis open mutants have increased k-on. We will compare the affinity of isolated I domains and intact alpha/beta heterodimers to test the hypothesis that some ligands bind both to the I domain and the 13-propeller domain while others bind only to the I domain. We will test the ability of isolated, high affinity I domains to block adhesive functions of leukocytes. We will examine binding of the alpha I domain to domain 3 of ICAM- I with co-crystal structures, and compare to the structure of the open mutant aM I domain in the absence of ligand. To characterize alphaX and beta2 stalk regions, we will map residues that restrain alphaXbeta2 in an inactive conformation. We will test expression of alphaM subunit C-terminal fragments, define the boundaries of domains recognized by antibodies, and test such fragments for crystallization.

R01CA031799
SPRINGER, TIMOTHY A

Time
1981-07-01
2006-11-30
Funded by the NIH National Center for Advancing Translational Sciences through its Clinical and Translational Science Awards Program, grant number UL1TR002541.