?(Fig.4)4) (17). IAP also enhances IL-2 production in Jurkat cells, an apparently integrin-independent function of IAP. Nonetheless, costimulation by IAP ligation requires cell adhesion. IAP costimulation does not require CD28. Furthermore, anti-IAP, but not anti-CD28, synergizes with suboptimal anti-CD3 to enhance tyrosine phosphorylation of the CD3 chain and the T cellCspecific tyrosine kinase Zap70. Ligation of human IAP transfected into the hemoglobin-specific 3.L2 murine T cell hybridoma costimulates activation for IL-2 secretion both with anti-CD3 and with antigenic peptides on antigen-presenting cells (APCs). Moreover, ligation of IAP but not CD28 can convert antagonist peptides into agonists in 3.L2 cells. Using costimulation by IAP ligation as an assay to analyze the structureCfunction relationships in IAP signaling, we find that both the extracellular and multiply membrane-spanning domains of IAP are necessary for synergy with the antigen receptor, but the alternatively spliced cytoplasmic tails are not. These data demonstrate that IAP ligation initiates an adhesiondependent costimulatory pathway distinct from CD28. We hypothesize that anti-IAP generates the costimulatory signal because it modulates interactions of the IgV domain name with other plasma membrane molecules; this in turn activates effector functions of the multiply membrane-spanning domain name of IAP. This model may have general significance for how IAP functions in cell activation. Integrin-associated protein (IAP)1 is usually CCT241736 a 50-kD highly hydrophobic cell surface glycoprotein that was originally copurified with the v3 vitronectin receptor from placenta (1) CCT241736 and later shown to be the antigen recognized by CD47specific mAb (2). Abs that recognize IAP inhibit some 3 integrin-mediated functions, including binding of vitronectin coated beads to cells, PMN activation by and chemotaxis to ArgCGlyCAsp (RGD)-made up of ligands, and endothelial [Ca2+]i increase during adhesion to fibronectin or vitronectin (3, 4). IAP has a broader cellular distribution than 3 integrins, suggesting that it may have functions other than those associated with 3. Recently, IAP has been shown to have a role in PMN migration across both endothelial and epithelial barriers (5, 6) and to bind the large multifunctional glycoprotein ILF3 thrombospondin (7), all functions without an obvious role for v3. Molecular cloning of IAP cDNAs from mouse and human revealed that it is an unusual Ig family member, with an Ig variable (IgV)- like amino terminal extracellular domain name, a domain name made up of multiple membrane spanning segments, and a short cytoplasmic tail (CT) with four alternatively CCT241736 spliced forms (8, 9). This three-domain structure raises the possibility that each domain name plays a discrete role in IAP function, but nothing is known about structureCfunction relationships of IAP. The ubiquity of IAP expression on continuous cell lines has hampered a systematic approach to this question. Although IAP is usually highly expressed on peripheral T lymphocytes, which express little if any v3, its function in these cells is not known. A potential role in T cell costimulation has been suggested by recent experiments (10). CCT241736 Whereas definitions of costimulation vary, in this work we have defined a costimulatory molecule as one that enhances T cell activation in response to a suboptimal antigen receptorCinitiated signal. Studies using mAbs directed against potential receptors for the costimulatory signal have identified more than 20 different T cell surface receptors, including multiple adhesion molecules, which can augment lymphocyte mitogenesis initiated by TCR engagement (11). The costimulatory receptors not only strengthen the adhesion between the antigen-responsive T cell and the APC, but also deliver crucial costimulatory signals to facilitate cytokine production and clonal expansion (12C14). Although CCT241736 CD28 is the most intensively studied costimulatory receptor, even in this case, the specific molecular events induced by ligation of CD28 required for costimulation remain uncertain. Although they retain the ability to bind to the MHC of the APC, antigenic peptides.