Fig. 5: JM and CT combine to regulate KD.

a Kinase activity is controlled by both JM and CT. KD^pY1, JM-KD^pY1, KD^pY1-CT^C1, and JM-KD^pY1-CT^C1, were incubated with kinase-dead JM-KD^K518I-CT^C1 in a 1:1000 ratio in the presence of ATP/Mg²⁺ and quenched with EDTA at different time points as indicated. The phosphorylation of JM-KD^K518I-CT^C1 was measured using a pY99 antibody. Densitometric line graph represents 2 independent experiments. The mean of the two replicates (shown as points) is represented by the colored lines. b Phosphorylation of CT (pCT^C1) reduces its binding to KD with higher phosphorylation order. GST-CT^C1 was phosphorylated by JM-KD^WT-CT^C1 and used for a GST pulldown assay with different phosphorylated JM-KD (JM-KD^pY1 – JM-KD^pY6 and wild type JM-KD). c GST-FRS2 PTB ___domain was used precipitate the following His-tagged constructs representing the mono-phosphorylated isoforms of FGFR2IIIb; JM-KD^pY1-CT^C1, JM-KD^pY1-CT^C2and JM-KD^pY1-CT^C3. The presence of the intact CT in the C1 isoform inhibits the interaction of FRS2 with its cognate site on JM. Densitometric bar graph represents 3 independent experiments. The error bars are presented as the standard deviation. d BLI measurement of GST-FRS2 PTB binding to JM-KD^pY1-CT^C1, JM-KD^pY1-CT^C2 and JM-KD^pY1-CT^C3. The GST-PTB ___domain from FRS2 was immobilized on the sensor and was exposed to 2.6 µM of the cytoplasmic region of each of the FGFR2 isoforms. After 300 s the chip was washed. The sensorgrams clearly show that over the time course up to 300 s (prior to the washing step; dotted line), in the absence of CT (C3 isoform) a significantly increased amount of FGFR2 protein binds to the PTB ___domain compared with the C1 and C2 isoforms. e The FGFR2 isoforms (JM-KD^pY1-CT^C1, JM-KD^pY1-CT^C2 and JM-KD^pY1-CT^C3) were incubated with FRS2 protein in a 1:100 ratio in the presence of ATP/Mg²⁺ and quenched at different time points as indicated. The phosphorylation of FRS2 was measured using an anti-pFRS2 (Y436) antibody. f. i: In the absence of stimulation the unphosphorylated FGFR2 (light blue, JM light blue line, CT red line) can exist as a monomer freely diffusing through the plasma membrane. ii: Random collision of FGFR2 results in dimer formation. Dimeric GRB2 (orange) is recruited via a proline-rich sequence on CT into a heterotetramer³⁰. This stabilizes the mono-phosphorylated active A-loop (green line) tyrosine residues (red circles) on KD, but signaling is stalled by the presence of GRB2 on CT. iii: the mono-phosphorylated KD also provides a strong binding site for CT. CT-KD^pY1 interaction results in the release of GRB2. The presence of CT on the KD prevents JM-mediated formation of asymmetric dimer and hence aberrant up-regulation. Active intermediate states ii: and iii: are in equilibrium, the concentration of the states is dependent on GRB2 concentration and the ability of GRB2 to compete with the intramolecular interaction with KD for binding to CT. iv: Binding of extracellular growth factor co-localizes two receptors into the active, asymmetric dimeric conformation. This is sustained by the interaction of JM from the enzyme-like receptor (dark blue) with KD of the substrate-like receptor (light blue). The sequence on JM which binds to KD (light blue thick line) is immediately proximal to a sequence (dark blue thick line) which binds in an independent interaction to CT of the enzyme-like receptor. This binding site (red thick line) includes the proline-rich motif that recognizes a site on KD and GRB2. Thus, JM-CT interaction blocks auto-inhibition and GRB2 recruitment. This ensures that the active state is prolonged. v: Prolonged activity of the dynamic asymmetric dimer results in increasing phosphorylation of KD and CT. As the pY burden increases the dimerization affinity between KDs reduces until they fully dissociate. The phosphorylated KD abrogates the inhibitory intramolecular binding of CT and the recruitment of GRB2. The receptor is therefore available for recruitment of downstream effector proteins (magenta). In the presence of growth factor the receptor would be expected to prevail as a dimer, however on dissociation of the growth factor the fully phosphorylated form may be expected to be a monomer.