Fibroblast growth factor receptors (FGFRs) initiate signal transduction via the RAS/MAPK pathway by their tyrosine-kinase activation known to determine cell-growth, tissue differentiation and apoptosis. Recently, many missense mutations have been reported for FGFR3, but we only know the functional effect for a handful of them. Some of these mutations result in aberrant FGFR3 signaling and are associated with various genetic disorders and oncogenic conditions. Here we employed micropatterned surfaces to specifically enrich fluorophore-tagged FGFR3 (mGFP-FGFR3) in certain areas of the plasma membrane of living cells. Receptor activation was then quantified via the recruitment of the downstream signal transducer GRB2 tagged with mScarlet (GRB2-mScarlet) to FGFR3 patterns. With this system, we tested the activation of FGFR3 upon ligand addition (fgf1 and fgf2) in the wildtype (WT), as well as in different FGFR3 mutants associated with congenital disorders (G380R, Y373C, K650Q, K650E). Our data showed that the addition of ligands increased GRB2 recruitment to WT FGFR3, with fgf1 having a stronger effect than fgf2. For all mutants, we found an increased basal receptor activity, and only for two of the four mutants (G380R and K650Q), activity was further increased upon ligand addition. Compared to previous reports, two mutant receptors (K650Q and K650E) had either an unexpectedly high or low activation state, respectively. This may be explained by the different receptor populations probed, since the micropatterning method specifically reports on signaling events at the plasma membrane.
Delayed fatherhood results in a higher risk to inherit a new germline mutation that might result in a congenital disorder in the offspring. In particular, some FGFR3 mutations increase in frequency with age, but there are still a large number of uncharacterized FGFR3 mutations that could be expanding in the male germline with potentially early or late-onset effects in the offspring. Here, we investigated the mutation frequency in the DNA of human testis and sperm and the activation state of the expressed mutant protein of eight different FGFR3 variants categorized by ClinVar as deleterious, benign, or not reported. Overall, the ligand-independent activation of the mutant protein resulted in a increased number of mutant sperm; although, strong activating mutations did not necessarily result in the highest frequencies. Moreover, only two mutants c.952G>A and c.1620C>A showed an increase with the donor's age; the latter also forming larger clonal expansions in the testis. We also showed that the prediction of deleteriousness of a mutation is not always accurate, and similar in silico scores can reflect either a gain-of-function or loss-of-function. Our approach led to the discovery of two novel variants c.1261G>A and c.952G>A to have promiscuous FGFR3 activation and increased mutation frequencies in the male germline. The large fraction of donors with mutations suggests a high de novo rate potentially explained by a selective advantage before the maturation of the male germline. This sequence-function study provides important data for the evaluation and interpretation of variants with relevant clinical implications
The results indicate that the evaporation rate is determined by defects in the wax ester rich TFLL and its coverage on the ocular surface. Meanwhile in the lungs, the pulmonary surfactant regulates the molecular transfer of oxygen to our body. The key protein governing oxygen transport through the pulmonary surfactant is SP-B. Based on molecular simulations (J. Liekkinen et al., J Mol Biol 432, 3251 (2020)) we discuss how the ring-like oligomeric SP-B complex binds to a pulmonary surfactant membrane through specific lipidprotein interactions and how SP-B reorganizes its lipid environment to modulate the pulmonary surfactant structure and function. The results suggest mechanisms that facilitate lipid and oxygen transfer in the pulmonary surfactant. In both of the two cases, we also discuss non-equilibrium behavior predicted by simulations.
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