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The composition of proteins recruited to the cytoplasmic domains of the integrins is influenced by the integrin heterodimer, the ECM and the cell type. In addition, the growth factor environment plays an important role. Integrins and growth factor or cytokine receptors can cooperate to provide specificity in signaling processes. They can act independently of each other, but the ECM and soluble factors, such as growth factors, hormones or cytokines, can also simultaneously activate integrins and receptors, signal synergistically and lead to the regulation of complex events. Adhesion to matrix molecules can influence growth factor receptors and vice versa. A precise spatial and temporal organization of the signals is very important for the resulting downstream effects. The interaction between integrins and the ECM is rather stable and therefore the signals from the microenvironment are sustained over a period of time. In contrast the diffusible growth factors and cytokines signal in a more temporal manner [108].

Impaired crosstalk between integrin and growth factor signaling results in diseases, for instance cancer. Cancer is characterized by missregulation of growth factor signaling and tumour cells can be independent from integrins leading to increased proliferation and/or migration [109; 110]. Loss of integrin mediated adhesion is one step in the development of metastases. On the contrary, cells can also upregulate integrins during tumour progression to enhance downstream signals [111].

There are several ways, in which growth factors can influence integrin function. Growth factor signaling can for instance change the expression level of certain integrins or integrin-binding proteins. TGF1 uses this mechanism to increase the

expression of the integrins 1 and 5 in endothelial cells [112]. A further possibility

how growth factor signaling interferes with integrin signaling is by influencing integrin conformation and by this the ligand affinity. They control the activation status of integrin associated signaling proteins like FAK, Src and PI3K and/or regulate downstream signaling effectors such as ERK, Akt, JNK and RhoGTPases (Figure 11A). For example, ERK activation resulting from cooperative integrin and growth factor signaling is increased in intensity and duration [77].

Chemokine-activated G-protein coupled receptors (GPCR) signal via PI3-kinase and phospholipase C (PLC) to increase integrin affinity. This represents a form of integrin inside-out signaling and is especially important for immune cells. Activation of the T cell antigen receptor upon antigen binding increases integrin activation in a PLC1-

and Rap1-dependent manner (Figure 11B) [113; 114]. Similar processes can activate integrins on platelets in the clotting cascade [115].

Figure 11: Signaling pathways resulting from crosstalk of integrins and growth factors. (A) Growth factor signaling influences integrin signaling in concert with the composition and the mechanical properties of the ECM [77]. (B) Signaling pathways leading to integrin activation in lymphocytes. Activation of a variety of receptors such as chemokine-activated G-protein coupled receptors or T cell receptors leads to recruitment of GTP-bound Rap1 and talin binding to the integrin tails, which results in activation of the integrins [114].

Conversely, integrins also have an impact on growth factor signaling. They can affect the cellular localization, posttranslational modification, expression of the receptors or the growth factors themselves. Integrins can also regulate receptor activation prior to ligand binding. Some integrins control for instance TGF signaling via binding to

latency-associated peptide [116]. Several receptor tyrosine kinases, such as PDGFR (platelet-derived growth factor receptor), EGFR (epidermal growth factor receptor), VEGFR (vascular endothelial growth factor receptor) and HGFR (hepatocyte growth factor receptor), can be activated simply by integrin mediated cell adhesion [117]. Most of these interactions are likely to be indirect.

An example for the crosstalk between integrins and RTKs is the cooperation of integrin 1 and EGFR in several cell types. Both activate ERK and Akt pathways in

order to regulate diverse cellular aspects, including cell cycle progression [118]. Integrins regulate proliferation via Src signaling and also in this process growth factor receptors contribute to the downstream effects. After ligand binding a fraction of EGFR is associated with integrin 1 in a complex with Src and p130Cas. This

complex is required for phosphorylation and activation of the EGF receptor [119].

Remarkably, this complex results in a distinct EGFR phosphorylation pattern, which is different from the phosphorylation pattern induced by EGF binding. This suggests additional functions of EGFR apart from the ones downstream of the growth factor. Integrin-mediated adhesion also controls interferon responses in a protein kinase C

dependent manner. For instance, STAT1 (signal transducer and activator of transcription) signaling in response to interferon is much stronger in adherent cells

than in cells in suspension [120].

Another example, in which integrins and growth factors collaborate, is the crosstalk between the integrin V3 and the M-CSF (macrophage colony stimulating factor)

receptor c-Fms in osteoclasts and their precursors. Signals from both receptors are required to regulate the osteoclast cytoskeleton, osteoclast differentiation as well as the proliferation of osteoclast precursors. Ligand binding by the integrin and M-CSF exposure simultaneously lead to a prolonged ERK signal [121]. The short-term effect of ERK activation is stimulation of precursor proliferation. Long duration of the signal induces for instance the expression of the transcription factor c-Fos and its nuclear translocation. This in turn promotes the expression of nuclear factors for activation of T cells (NFATs) that are central in osteoclast differentiation by upregulation of certain genes (Figure 12) [122]. The activation of c-Fms also leads to signaling through PI3K, which further activates Akt. Both proteins are also downstream targets of integrin signaling and therefore represent a further intersection of integrin-growth factor crosstalk [123]. Furthermore, integrin V3 and c-Fms act via the non-receptor

tyrosine kinase Syk. Activation of the V3 heterodimer is followed by the activation

of Src, probably by autophosphorylation. Src phosphorylates DAP12 (DNAX- activating protein of 12 kDa) and FcR (fragment (crystallisable) receptor ), which

are adaptor molecules containing an immunoreceptor tyrosine-based activation motif (ITAM). The phosphorylation of ITAM provides a binding site for Syk and results in the recruitment of Syk to the cytoplasmic domain of the integrin subunit. Syk

subsequently interacts with Src and becomes activated. Downstream of Syk the Rac GEF Vav3 is activated. The GTP-bound form of Rac finally initiates cytoskeleton reorganization [123; 124]. Binding of M-CSF to its receptor on the other hand leads to autophosphorylation of the receptor generating a binding site for Src at tyrosine 559. This interaction also results in Src autophosphorylation and the phosphorylation of DAP12. The subsequent recruitment and activation of Syk again lead to cytoskeletal rearrangement via Vav3 and Rac (Figure 12) [123; 125].

Figure 12: Scheme of the crosstalk between integrin V3 and the M-CSF receptor c-Fms. ECM binding and M-CSF exposure together lead to the activation of a signaling pathway including ERK, which leads to differentiation via the nuclear proteins c-Fos and NFAT. Ligand bound integrin V3 and activated c-Fms also result in activation of the RhoGTPase Rac via Src, Syk and Vav3 [123].