␣ 4 ␤ 1 integrin (VLA-4) appears to be unique among the leukocyte integrins in that it can initiate the adhesion of circulating lymphocytes without cellular activation. It is not known how lymphocytes or other cell types maintain constitutive levels of ␣ 4 ␤ 1 integrin activity. The current report describes a monoclonal antibody, 15/7, that recognizes a high affinity or ligand-occupied conformation of ␤ 1 integrin. Studies with 15/7 revealed that ␣ 4 ␤ 1 integrin-dependent adhesion of leukocytic cell lines is mediated by a population of low affinity receptors that is conformationally responsive to ligand; the 15/7 epitope could be induced by nanomolar concentrations of soluble VCAM-1 or by micromolar concentrations of a peptide derived from the type III connecting segment domain of fibronectin (as ligands for ␣ 4 ␤ 1 integrin). The same receptors were also responsive to adhesion activating reagents, such as Mn 2؉ , activating anti-␤ 1 integrin antibodies, and phorbol myristate acetate, which induced the 15/7 epitope directly and/or decreased the concentration of ligand required for epitope induction. In addition to the responsive receptor pool, cells expressed a second population of ␣ 4 ␤ 1 integrin that was conformationally restrained, failing to respond to ligand or to any of the activating reagents. The relative size of the responsive and inactive receptor pools, as well as the affinity of the responsive receptors, represented a stable phenotype of different cell types and played important roles in defining the cells' adhesive capacity and ligand specificity. Similar receptor populations were measured on lymphocyte subsets in whole blood. These studies provide insight into how cells maintain different constitutive levels of ␣ 4 ␤ 1 integrin activity, and how the activity of ␤ 1 integrin can be modulated by activators of cell adhesion.Integrins are heterodimeric adhesion molecules that contribute to the specificity of cellular interactions through the recognition of numerous matrix and cell-associated ligands (1). Importantly, the ligand binding activity of integrins can be modulated rapidly, allowing cells to specify the timing and location of integrin-mediated adhesive interactions. On circulating leukocytes, for example, the ␤ 2 integrins LFA-1 and Mac-1 are thought to be activated by site-specific factors (cytokines or other adhesive interactions) during transient cell interactions with the vascular wall; the receptors then establish firm adhesive contacts and mediate leukocyte extravasation (2, 3). Likewise, platelets are stimulated at sites of vascular injury allowing ␣II b ␤ 3 integrin to bind to fibrinogen and initiate thrombosis (4).The regulation of ␣ 4 integrin activity on circulating immune cells appears to be different from that of the other leukocyte integrins described above. ␣ 4 ␤ 1 and ␣ 4 ␤ 7 integrin can mediate leukocyte adhesion to their endothelial ligands VCAM-1 (5) and MAdCAM-1 (6), respectively, without cellular activation, and can do so even in the presence of the shear forces encount...

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Abstract. Transferrin receptors in detergent extracts of subcellular membrane fractions prepared from 3T3-L1 adipocytes were measured by a binding assay. There was a small but significant increase (1.2-fold) in the amount of receptor in a crude plasma membrane fraction and a 40% decrease in the number of transferrin receptors in microsomal membranes prepared from insulin-treated cells, when compared with corresponding fractions from control cells. Intracellular vesicles containing insulin-responsive glucose transporters (GT) have been isolated by immunoadsorption from the microsomal fraction (Biber, J. W., and G. E. Lienhard. 1986. J. Biol. Chem. 261:16180-16184). All of the transferrin receptors in this fraction were localized in these vesicles; however, because the GT vesicles contain '~30-fold fewer transferrin receptors than GT, on the average only one vesicle in three contains a transferrin receptor.The binding of ~25I-pentamannose 6-phosphate BSA to 3T3-L1 adipocytes at 4°C was used to monitor surface insulin-like growth factor II (IGF-II)/mannose 6-phosphate receptors. Exposure of cells to insulin at 37°C for 5 min resulted in a 2.5-4.5-fold increase in surface receptors. There was a corresponding 20% decrease in the amount of IGF-II receptors in the microsomal membranes prepared from insulin-treated cells, as assayed by immunoblotting. Moreover, the IGF-II receptors and GT were located in the same intracellular vesicles, since antibodies to the carboxyterminal peptide of either protein immunoadsorbed vesicles containing 70-95 % of both proteins initially present in the microsomal fraction. In conjunction with other studies, these results indicate that in 3T3-L1 adipocytes, three membrane proteins (the GT, the transferrin receptor, and the IGF-II receptor) respond similarly to insulin, by redistributing to the surface from intracellular compartment(s) in which they are colocalized.

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