These results address three issues in integrin-mediated signaling: 1) Is there a proportional dose-response relationship in signal initiation, and if so, how do intracellular processes read the extracellular signaling levels? 2) Can different integrin-ligand interactions elicit different intracellular signals? And 3) what are the contributions of integrin clustering, ligand occupancy, and integrin tethering to integrin-mediated signals? An experimental paradigm was developed that uses a kinetic approach in combination with technologies to quantify both the strength of adhesion and the number of ligated receptors to relate the proportion of integrin bound to the level of signaling. The analysis is focused on the phosphorylation of tyrosines Y397, Y407, Y577, Y861, and Y925. This may not represent the full output of α5β1-mediated signals.
If the binding of integrin to its ligand functions analogously to cytokine receptors or to receptor-tyrosine-kinases, then an increase in the number of bound receptors should generate a proportional increase in the level of downstream signals. As the signals proceed further downstream, some amplification of the signal is expected to generate a final all-or-none response (Ferrell, 1998
). For the receptor tyrosine kinases, ligand binding results in the autophorylation of sites in the cytoplasmic domain that in turn provide sites for the binding of downstream effectors (Livneh et al., 1986
). The total level of phosphorylation is proportional to the ligand concentration and the number of ligands bound. In this case and with other receptor systems, the internal cell read-out is provided by phosphorylation of the receptor itself or its associated proteins (Hunter and Cooper, 1981
; Livneh et al., 1986
). For α5β1 integrin a prime candidate for providing this read-out function is FAK. Previous analyses have identified FAK as an early target in integrin signaling (Guan et al., 1991
; Kornberg et al., 1991
; Schlaepfer et al., 1999
) and have identified Y397 as a critical first step in the process (Owen et al., 1999
The data demonstrate an increase in the level of both Y397 and Y861 phosphorylation that directly parallels the increase in the number of α5β1-fibronectin bonds. The relative number of α5β1 fibronectin bonds was correlated to both the proportion of α5 and β1 subunits that could be cross-linked and to the overall strength of adhesion measured using the spinning disk. Taken together, these results suggest that the level of phosphorylation of FAK Y397 is a direct consequence of the number of (mechanically strong) α5β1-fibronectin bonds. This provides a means for the cell to measure the mechanical and biochemical environment. The reason that we do not include Y861 as a primary measure of α5β1 bonds is that the coupling between Y861 phosphorylation and the number of bonds was weaker in other cell types: including, chicken embryo fibroblasts (Datta et al., 2001
), IMR90 human fibroblasts, and HOS osteosarcoma cells, and because it responded to alternate signaling mechanisms. When HT1080 cells were plated on fibronectin, both tethering and clustering occur as part of the normal process. Thus, we cannot exclude clustering as an explanation for the increase in Y861 phosphorylation in those experiments.
The finding that the levels of phosphorylation of Y397 were directly proportional to the number of mechanically functional α5β1-fibronectin bonds provides a means for the cell to respond to integrin-mediated adhesion to a substrate. It is particularly significant that Y397 is the responding site because this site is central to the formation of complexes based on the binding of SH2 domains to its phosphorylated form. The SH2 domains of src, PI3-kinase, PLCγ, Grb7, and Shc have been reported to bind pY397 providing many potential downstream signaling consequences (Schaller et al., 1994
; Schlaepfer and Hunter, 1997
; Han and Guan, 1999
; Reiske et al., 1999
; Zhang et al., 1999
Most current integrin signaling data has been interpreted in the context of the integrin-clustering model. The plating of fibroblasts on a fibronectin-coated surface induces not only the binding of α5β1 to fibronectin, but also the progressive organization of α5β1 into focal contacts and focal adhesions during the process of cell spreading (Singer et al., 1988
; Pankov et al., 2000
). Because the experiments described here do involve the plating of cells on a fibronectin substrate, a reorganization of integrin into focal contacts and focal adhesion did occur. Therefore, these data do not exclude a role for clustering in integrin-mediated signals, including the phosphorylation of Y397. The failure of clustering to stimulate Y397 phosphorylation in the absence of adhesion demonstrates that clustering alone is not a sufficient explanation for integrin-mediated signaling. Furthermore, the demonstrations that the same antibody and ligand combinations that elicited Y861 but not Y397 phosphorylation in the clustering protocol could stimulate phosphorylation Y397 when they were adsorbed to the substrate. This reinforces the conclusion that it is the tethering of α5β1 to the substrate, rather than its clustering, that provides the signal for phosphorylation of Y397.
In many transmembrane receptor signaling systems the role of the ligand is primarily to cluster or dimerize the receptor (Schlessinger, 2000
). As long as the dimerization takes place, it does not matter whether the receptor is occupied or not. In the experiments described here, neither clustering integrins with an activating mAb to β1 integrin nor addition of a ligand and clustering through that ligand was able to induce the phosphorylation of Y397. Also, the use of antibodies bound to the substrate, in the absence of ligand, was still able to stimulate Y397 phosphorylation. These data imply that for stimulation of Y397 phosphorylation, it does not matter whether α5β1 is occupied by ligand or not, only that it is tethered to the substrate. In contrast to these results, there are other reports that suggest that ligand occupation itself could affect signaling; however, different signaling end points were used for those experiments and analysis of receptor occupancy effects was not the author's primary objective. Miyamoto et al.
) analyzed the recruitment of α-actinin, vinculin, and talin to beads coated with adhesion-blocking antibodies or noninhibitory integrin antibodies. In the presence of inhibitors, including cytochalasin D, these proteins were recruited to the bead sites only by the inhibitory antibodies. This was interpreted as a requirement for ligand occupancy for their recruitment. However, these antibodies also differ in other properties such as location of their binding epitope and ligand-binding affinity, which could influence the recruitment. Arthur et al.
) report the downregulation of RhoA activity in src-reconstituted SYF mouse fibroblasts in response to the addition of soluble RGD. But, the actual mechanism and possible receptors involved was not defined. Thus, it is possible that ligand occupancy can be detected by the intracellular signaling processes, but a clear answer is not yet available.
The data demonstrate a requirement for the tethering of α5β1 to substrate for the induction of phosphorylation or the formation of phosphorylated complexes, involving FAK Y397. Neither the clustering of α5β1, nor the binding of ligand to α5β1, was able to substitute for this requirement. Thus, this constitutes a novel mode for the transmission of signals from the extracellular environment across the plasma membrane to link with intracellular signaling pathways. The clustering of α5β1 and the tethering of α5β1 led to different patterns of FAK phosphorylation, raising the possibility that the downstream signals may depend, not only on the particular cell type, but also on the way in which the signal is delivered.