Affinity Modulation of Integrin t 5 / 31 : Regulation of the Functional Response by Soluble Fibronectin

We report that a E1 integrin (o~5fll) can exist in different affinity states for its soluble ligand, fibronectin. The ot5fll expressed by the erythroleukemic cell line K562 binds soluble fibronectin with low affinity (Ka > 1 #M), but is induced to bind it with 20-fold higher affinity (Kd-54 nM) in the presence of the anti-ill mAb 8A2. This activation seems to be due to direct antibody-induced change in the receptor that does not require intracellular signaling, and is a plausible basis for the 8A2-induced enhancement of ill-dependent adhesion to fibronectin and other immobilized ligands (Kovach, N. L., T. M. Carlos, E. Yee, and J. M. Harlan. 1992. J. Cell BioL 116: 499-509). Fab fragments of 8A2 bind with higher affinity to ot5fll receptor that is occupied by the GRGDSP peptide ligand suggesting that the antibody functions by stabilizing a high affinity (occupied) conformer of the receptor. A functional consequence of the affinity modulation is that soluble fibronectin (at physiological concentrations) occupies the high affinity receptors, and so becomes an effective inhibitor of adhesion to immobilized fibronectin. In contrast, the majority of low affinity receptors remain unoccupied and are still able to mediate cellular adhesion. C eLL adhesion is controlled by interactions between adhesion receptors and their ligands. Changing the repertoire of adhesive receptors, by either modifying biosynthetic patterns (16, 39, 45, 47) or by receptor translocations (8, 35), are strategies to modify cell adhesion. The function of adhesion receptors can also be changed by structural alterations, by changes in mRNA splicing (5, 10, 30, 41, 54), proteolytic cleavage (31), or variations in glycosylation (17). Finally, cellular adhesion can be rapidly regulated through reversible modulation of receptor function, and this mechanism is a key feature of the integrin family of adhesion receptors (4, 15, 42, 51, 52). The most widespread integrins are the/31 subfamily (VLA family) (27-29), and there is dynamic regulation of fll integrin-mediated adhesion. T lymphocytes and T lymphoid cell lines, activated either with phorbol esters, by crosslinking CD3, or with activating combinations of anti-CD2 mAbs, exhibit increased ill-dependent adhesion to immobilized ligands (51, 52). This occurs without alterations in their repertoire of integrins. Cellular adhesion to immobilized ligand is a complex multi-step process, involving initial receptor-ligand interaction, followed by strengthening of adhesion through organization of the cytoskeleton, focal contact formation, and cell spreading (11, 55). Cells could enhance adhesion by the facilitation of any of these steps. For example, Danilov et al. (14) found that phorbol esters enhanced the adhesion of CHO cells to immobilized fibronectin without any detectable change in the affinity of their fibronectin receptors for soluble ligand. Alternatively, the ligand binding affinity of certain integrins can be rapidly altered. For example, platelet receptor allbfl3 (or GPIIbHIa) exists in at least two affinity states for its soluble ligand, fibrinogen (7, 33, 50), which appear to be a result of different conformations of the receptor (13, 40). Conversion from low to high affinity binding of fibrinogen can be induced by treating the platelets with agonists such as phorbol esters, ADP, and thrombin (43, 44), and by certain mAbs directed against the f13 chain (40). Interconversion between low and high affinity fihrinogen binding states also occurs with otMff2 (4). Direct demonstration of affinity modulation of fll integrins has not been reported. However, it is known that soluble fibronectin binds with moderate affinity to E1 integrins expressed by cell lines such as CliO cells (K~ = 100 nM) (14) and BHK cells (Kd = 800 riM) (1). Several anti-B1 mABs which enhance/31 integrin-dependent cellular adhesion have been described (6, 32, 38, 53). In this paper we have used one of these antibodies (8A2, reference 32), to investigate affinity modulation of the fibronectin receptor, ot5fll. We show that the a5fll expressed by the erythroleukemic cell line K562 binds soluble fibronectin with low affinity which can be increased 20-fold by 8A2. The alteration in affinity occurs in the presence of inhibitors of intracellular signaling, and it appears that the antibody functions by directly inducing a high affinity conformation of the © The Rockefeller University Press, 0021-9525/93/04/155/8 $2.00 The Journal of Cell Biology, Volume 121, Number 1, April 1993 155-162 155 on O cber 8, 2017 jcb.rress. org D ow nladed fom receptor. Finally, the biologic consequences of affinity modulation of a5B1 were found to depend on the ambient soluble fibronectin concentration. Materials and Methods Antibodies and Reagents Production and initial characterization of the murine anti-human/31 mAb 8A2 (IgGl-r isotype) has been described (32). The rat anti-human ~5 mAb BIIG2 and rat anti-human/~1 mAb AIIB2 (23) were used as dilutions of ascites. The murine anti-human a4 mAb HP2/1 (49) was used as dilutions of tissue culture supernatant. The murine anti-human/~1 mAbs AlAS and TS2/16 (25) were used as dilutions of ascites. The murine anti-human fibronectin mAb Fnl.ll was produced in our laboratory as described (9), and used as dilutions of ascites. The murine anti-human HLA class I mAb W6/32 was obtained from the American Type Culture Collection (Rockville, MD) and used as dilutions of purified IgG. The peptide GRGDSP was obtained from Peninsula Laboratories (Belmont, CA) and the peptide GRGESP was synthesized in the Scripps Research Institute core facility (La Jolla, CA). Glucose and 2-deoxyglucose were purchased from Sigma (St. Louis, MO) and sodium azide from Fisher Scientific Co. (Pittsburgh, PA).