Integrin αIIbβ3 mediates platelet aggregation and “outside-in” signaling. It is regulated by changes in receptor conformation and affinity and/or by lateral diffusion and receptor clustering. To document the relative contributions of conformation and clustering to αIIbβ3 function, αIIb was fused at its cytoplasmic tail to one or two FKBP12 repeats (FKBP). These modified αIIb subunits were expressed with β3 in CHO cells, and the heterodimers could be clustered into morphologically detectable oligomers upon addition of AP1510, a membrane-permeable, bivalent FKBP ligand. Integrin clustering by AP1510 caused binding of fibrinogen and a multivalent (but not monovalent) fibrinogen-mimetic antibody. However, ligand binding due to clustering was only 25–50% of that observed when αIIbβ3 affinity was increased by an activating antibody or an activating mutation. The effects of integrin clustering and affinity modulation were additive, and clustering promoted irreversible ligand binding. Clustering of αIIbβ3 also promoted cell adhesion to fibrinogen or von Willebrand factor, but not as effectively as affinity modulation. However, clustering was sufficient to trigger fibrinogen-independent tyrosine phosphorylation of pp72Syk and fibrinogen-dependent phosphorylation of pp125FAK, even in non-adherent cells. Thus, receptor clustering and affinity modulation play complementary roles in αIIbβ3 function. Affinity modulation is the predominant regulator of ligand binding and cell adhesion, but clustering increases these responses further and triggers protein tyrosine phosphorylation, even in the absence of affinity modulation. Both affinity modulation and clustering may be needed for optimal function of αIIbβ3 in platelets.

Integrin ␣ V ␤ 3 mediates diverse responses in vascular cells, ranging from cell adhesion, migration, and proliferation to uptake of adenoviruses. However, the extent to which ␣ V ␤ 3 is regulated by changes in receptor conformation (affinity), receptor diffusion/clustering (avidity), or post-receptor events is unknown. Affinity regulation of the related integrin, ␣ IIb ␤ 3 , has been established using a monovalent ligand-mimetic antibody, PAC1 Fab. To determine the role of affinity modulation of ␣ V ␤ 3 , a novel monovalent ligand-mimetic antibody (WOW-1) was created by replacing the heavy chain hypervariable region 3 of PAC1 Fab with a single ␣ V integrin-binding domain from multivalent adenovirus penton base. Both WOW-1 Fab and penton base bound selectively to activated ␣ V ␤ 3 , but not to ␣ IIb ␤ 3 , in receptor and cell binding assays. ␣ V ␤ 3 affinity varied with the cell type. Unstimulated B-lymphoblastoid cells bound WOW-1 Fab poorly (apparent K d ‫؍‬ 2.4 M), but acute stimulation with phorbol 12-myristate 13-acetate increased receptor affinity >30-fold (K d ‫؍‬ 80 nM), with no change in receptor number. In contrast, ␣ V ␤ 3 in melanoma cells was constitutively active, but ligand binding could be suppressed by overexpression of ␤ 3 cytoplasmic tails. Up-regulation of ␣ V ␤ 3 affinity had functional consequences in that it increased cell adhesion and spreading and promoted adenovirus-mediated gene transfer. These studies establish that ␣ V ␤ 3 is subject to rapid regulated changes in affinity that influence the biological functions of this integrin.Integrins mediate cell adhesion and signaling during many developmental, physiological, and pathological processes (1-4). The ␤ 3 integrin family includes ␣ IIb ␤ 3 , often referred to as the fibrinogen receptor, and ␣ V ␤ 3 , the vitronectin receptor. ␣ IIb ␤ 3 is confined to megakaryocytes and platelets and is required for platelet aggregation through interactions with Arg-Gly-Asp (RGD)-containing adhesive ligands, including fibrinogen and von Willebrand factor (5). ␣ V ␤ 3 is more widely expressed in proliferating endothelial cells, arterial smooth muscle cells, osteoclasts, platelets, and certain subpopulations of leukocytes and tumor cells (6, 7). The list of cognate ligands for ␣ V ␤ 3 overlaps that for ␣ IIb ␤ 3 , but includes others, such as osteopontin, matrix metalloproteinase-2, and adenovirus penton base, which do not interact with ␣ IIb ␤ 3 (6, 8 -10). In the adult organism, ␣ V ␤ 3 has been implicated in processes ranging from wound healing to tumor angiogenesis (11), arterial restenosis (12), osteoporosis (13), tumor progression (14), and adenovirus internalization (8).One fundamental function of integrins is ligand binding, which in many cases is rapidly regulated by a process variously referred to as "integrin activation," "inside-out signaling," or "affinity/avidity modulation" (15-19). Integrin activation encompasses at least two events: 1) modulation of receptor affinity through conformational changes in the ␣␤ heterodimer and 2) modulation...

Aurora-A kinase (Aur-A) is a member of the serine/threonine kinase family that regulates the cell division process, and has recently been implicated in tumorigenesis. In this study, we identified an antigenic 9-amino-acid epitope (Aur-A(207-215): YLILEYAPL) derived from Aur-A capable of generating leukemia-reactive cytotoxic T lymphocytes (CTLs) in the context of HLA-A*0201. The synthetic peptide of this epitope appeared to be capable of binding to HLA-A*2402 as well as HLA-A*0201 molecules. Leukemia cell lines and freshly isolated leukemia cells, particularly chronic myelogenous leukemia (CML) cells, appeared to express Aur-A abundantly. Aur-A-specific CTLs were able to lyse human leukemia cell lines and freshly isolated leukemia cells, but not normal cells, in an HLA-A*0201-restricted manner. Importantly, Aur-A-specific CTLs were able to lyse CD34+ CML progenitor cells but did not show any cytotoxicity against normal CD34+ hematopoietic stem cells. The tetramer assay revealed that the Aur-A(207-215) epitope-specific CTL precursors are present in peripheral blood of HLA-A*0201-positive and HLA-A*2402-positive patients with leukemia, but not in healthy individuals. Our results indicate that cellular immunotherapy targeting Aur-A is a promising strategy for treatment of leukemia.

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