Indirect recruitment of the signalling adaptor Shc to the fibroblast growth factor receptor 2 (FGFR2).

The adaptor protein Shc (Src homology and collagen-containing protein) plays an important role in the activation of signalling pathways downstream of RTKs (receptor tyrosine kinases) regulating diverse cellular functions, such as differentiation, adhesion, migration and mitogenesis. Despite being phosphorylated downstream of members of the FGFR (fibroblast growth factor receptor) family, a direct interaction of Shc with this receptor family has not been described to date. Various studies have suggested potential binding sites for the Shc PTB domain (phosphotyrosine-binding domain) and/or the SH2 (Src homology 2) domain on FGFR1, but no interaction of full-length Shc with these sites has been reported in vivo. In the present study, we investigated the importance of the SH2 domain and the PTB domain in recruitment of Shc to FGFR2(IIIc) to characterize the interaction of these two proteins. Confocal microscopy revealed extensive co-localization of Shc with FGFR2. The PTB domain was identified as the critical component of Shc which mediates membrane localization. Results from FLIM (fluorescence lifetime imaging microscopy) revealed that the interaction between Shc and FGFR2 is indirect, suggesting that the adaptor protein forms part of a signalling complex containing the receptor. We identified the non-RTK Src as a protein which potentially mediates the formation of such a ternary complex. Although an interaction between Src and Shc has been described previously, in the present study we implicate the Shc SH2 domain as a novel mediator of this association. The recruitment of Shc to FGFR2 via an indirect mechanism provides new insight into the regulation of protein assembly and activation of various signalling pathways downstream of this RTK.

Extracellular point mutations in FGFR2 elicit unexpected changes in intracellular signalling.

An understanding of cellular signalling from a systems-based approach has to be robust to assess the effects of point mutations in component proteins. Outcomes of these perturbations should be predictable in terms of downstream response, otherwise a holistic interpretation of biological processes or disease states cannot be obtained. Two single, proximal point mutations (S252W and P253R) in the extracellular region of FGFR2 (fibroblast growth factor receptor 2) prolong growth factor engagement resulting in dramatically different intracellular phenotypes. Following ligand stimulation, the wild-type receptor undergoes rapid endocytosis into lysosomes, whereas (SW)FGFR2 (the S252W FGFR2 point mutation) and (PR)FGFR2 (the P253R FGFR2 point mutation) remain on the cell membrane for an extended period of time, modifying protein recruitment and elevating downstream ERK (extracellular-signal-regulated kinase) phosphorylation. FLIM (fluorescent lifetime imaging microscopy) reveals that direct interaction of FRS2 (FGFR substrate 2) with wild-type receptor occurs primarily at the vesicular membrane, whereas the interaction with the P253R receptor occurs exclusively at the plasma membrane. These observations suggest that the altered FRS2 recruitment by the mutant receptors results in an abnormal cellular signalling mechanism. In the present study these profound intracellular phenotypes resulting from extracellular receptor modification reveal a new level of complexity which will challenge a systems biology interpretation.

A phosphorylation-dependent gating mechanism controls the SH2 domain interactions of the Shc adaptor protein.

The Shc (Src homology collagen-like) adaptor protein plays a crucial role in linking stimulated receptors to mitogen-activated protein kinase activation through the formation of dynamic signalling complexes. Shc comprises an N-terminal phosphotyrosine binding (PTB) domain, a C-terminal Src homology 2 (SH2) domain and a central proline-rich collagen homology 1 domain. The latter domain contains three tyrosine residues that are known to become phosphorylated. We have expressed and purified the human p52Shc isoform and characterised its binding to different ligands. CD spectra revealed that some parts of the Shc protein are not fully folded, remaining largely unaffected by the binding of ligands. The PTB domain binds peptide and Ins-1,4,5-P(3) (but not Ins-1,3,5-P(3)) independently, suggesting two distinct sites of interaction. In the unphosphorylated Shc, the SH2 domain is non-functional. Ligand binding to the PTB domain does not affect this. However, phosphorylation of the three tyrosine residues promotes binding to the SH2 domain. Thus, Shc has an intrinsic phosphorylation-dependent gating mechanism where the SH2 domain adopts an open conformation only when tyrosine phosphorylation has occurred.

Extracellular point mutations in FGFR2 result in elevated ERK1/2 activation and perturbation of neuronal differentiation.

Two independent gain-of-function point mutations (S252W and P253R) in the extracellular region of the FGFR2 (fibroblast growth factor receptor 2) increase the binding affinity for the growth factor. The effect of this enhanced growth factor binding by these mutants is expected to be an increase in activation of regular signalling pathways from FGFR2 as a result of more receptors being engaged by ligand at any given time. Using PC12 (pheochromocytoma) cells as a model cell system we investigated the effect of these mutations on protein phosphorylation including the receptor, the activation of downstream signalling pathways and cell differentiation. Our results show that the effects of both of these extracellular mutations have unexpected intracellular phenotypes and cellular responses. Receptor phosphorylation was altered in both the ligand-stimulated and unstimulated states. The mutants also resulted in differential phosphorylation of a number of intracellular proteins. Both mutations resulted in enhanced ERK1/2 (extracellular-signalregulated kinase1/2) activation. Although ERK1/2 activation is believed to transduce signals resulting in cell differentiation, this response was abrogated in the cells expressing the mutant receptors. The results of the present study demonstrate that single extracellular point mutations in the FGFR2 have a profound effect on intracellular signalling and ultimately on cell fate.