Dimerization And Activation

• A N Plotnikov
• Dimerization Activation
• Joseph Schlessinger

Fibroblast growth factors (fgfs) are widely believed to activate their receptors by mediating receptor dimerization. Here we show, however, that the FGF receptors form dimers in the absence of ligand, and that these unliganded dimers are phosphorylated. We further show that ligand binding triggers structural changes in the FGFR dimers, which increase FGFR phosphorylation. The observed effects due to the ligands fgf1 and fgf2 are very different.

A N Plotnikov

Dimerization Activation

The fgf2-bound dimer structure ensures the smallest separation between the transmembrane (TM) domains and the highest possible phosphorylation, a conclusion that is supported by a strong correlation between TM helix separation in the dimer and kinase phosphorylation. The pathogenic A391E mutation in FGFR3 TM domain emulates the action of fgf2, trapping the FGFR3 dimer in its most active state. This study establishes the existence of multiple active ligand-bound states, and uncovers a novel molecular mechanism through which FGFR-linked pathologies can arise.

The fibroblast growth factor receptor (FGFR) family includes four receptors that bind 18 ligands called fibroblast growth factors, using heparin as a co-factor. These receptors play important roles in all cell types, but are best known for the critical role that they play in the development of the skeletal system. Many pathogenic mutations of FGFR genes are linked to skeletal, cranial and other developmental abnormalities in humans. Furthermore, FGFR overexpression and mutations have been reported in a variety of cancers,.FGF receptors are single-pass membrane proteins, with N-terminal extracellular (EC) domains consisting of three immunoglobulin-like subdomains (D1, D2 and D3), a transmembrane (TM) domain consisting of a single α-helix, and an intracellular (IC) region encompassing a tyrosine kinase domain.

Joseph Schlessinger

FGFRs transduce biochemical signals via lateral dimerization in the plasma membrane. Receptor dimerization is necessary for activation, as it brings the two tyrosine kinase domains into close proximity, allowing them to cross-phosphorylate each other on tyrosines in their activation loops. This activates the kinases, which then bind adaptor proteins and phosphorylate cytoplasmic substrates, triggering downstream signalling cascades that control cell growth and differentiation.High-resolution crystal structures of isolated FGFR EC domains in the presence of different fgfs have provided detailed views of ligand–receptor and receptor–receptor interactions in the EC portion, as well as the role of the co-factor heparin,. However, there is little mechanistic understanding of how conformational changes are transmitted from the EC domains through the TM domains to the kinase domains, in response to ligand binding. Different fgf ligands can elicit distinctly different biological responses, but the mechanism behind the specificity is unknown.

To gain insight into these issues, here we study the dimerization of FGFR1, FGFR2 and FGFR3, as well as the response of these receptors to the ligands fgf1 and fgf2. Our results show that ligand binding to unliganded FGFR dimers triggers a switch to ligand-specific configurations of the TM helices, which in turn increase receptor phosphorylation. We further show that a pathogenic FGFR mutant causes unregulated ligand-independent signalling by mimicking the most active ligand-bound configuration. ( a) Measured FRET in plasma membrane-derived vesicles, as a function of receptor concentration, for FGFR1 (black), FGFR2 (olive) and FGFR3 (red).

Every data point represents a single vesicle. ( b) The donor concentration is plotted as a function of the acceptor concentration, for each vesicle. ( c) Dimeric fraction as a function of total receptor concentrations. The experimentally determined dimeric fractions are binned and are shown with the symbols, along with the standard errors. Each bin contains between 5 and 50 experimental points.

The solid lines are the dimerization curves, plotted for the optimized dimerization parameters in. The dimeric receptor fraction as a function of receptor concentration is shown in. From this concentration dependence we obtained, by fitting, the two-dimensional dissociation constant K diss and the structural parameter ‘intrinsic FRET’, (refs,; ).

Intrinsic FRET does not depend on the dimerization propensities, and is directly related to the distance between the fluorescent proteins. As discussed below, measurements of intrinsic FRET allow us to capture structural changes that occur on the cytoplasmic side of the receptor on ligand binding to the extracellular domains. The values of the two-dimensional dissociation constants, K diss are 710, 111 and 24 μm −2 for FGFR1, FGFR2 and FGFR3, respectively, corresponding to dimerization free energies of −4.3±0.1, −5.4±0.1 and −6.3±0.1 kcal mol −1 (see equation (10) and; uncertainties are standard errors). The intrinsic FRET values for the unliganded FGFR1, FGFR2 and FGFR3 dimers are 0.66, 0.43 and 0.55, respectively.To evaluate the biological significance of the measured unliganded dimerization of the FGFRs, we note that physiological FGFR expression levels can be as high as ∼80 000 receptors per cell, corresponding to ∼80–100 receptors per μm 2 (ref.