Measurement of FGFR3 signaling at the cell membrane via total internal reflection fluorescence microscopy to compare the activation of FGFR3 mutants

Hartl, Ingrid; Brumovska, Veronika; Striedner, Yasmin; Yasari, Atena; Schütz, Gerhard J.; Sevcsik, Eva; Tiemann‐Boege, Irene

doi:10.1016/j.jbc.2022.102832

Cited by 2 publications

(12 citation statements)

References 60 publications

(116 reference statements)

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“…The second novel variant, c.1862G > T (p.R621L) showed a significantly decreased GRB2 recruitment compared to the WT receptor both in the nonliganded and the FGF1-activated state, similar to the established negative control ( Hartl et al 2023 ). This variant has never been described in the literature and has no associated syndromes.…”

Section: Discussionmentioning

confidence: 76%

“…Variant c.1138G > A (p.G380R) is considered a canonical PAE mutation, and causes 98% of the spontaneous achondroplasia (ACH) cases (MIM #100800) ( Shiang et al 1994 ; Bellus et al 1995 ). The mutation associated with ACH affects the transmembrane domain of FGFR3 and has been described to result in a higher signaling activity ( Naski et al 1996 ; Webster et al 1996 ; Hartl et al 2023 ). In particular, c.1138G > A was also shown to (i) increase in frequency in sperm of older donors ( Tiemann-Boege et al 2002 ), (ii) to form subclonal clusters in the testis ( Shinde et al 2013 ; Maher et al 2018 ), and (iii) to occur at a higher incidence in offspring of older fathers ( Risch et al 1987 ).…”

Section: Resultsmentioning

confidence: 99%

“…5a ) that allows the study of protein–protein interactions at the plasma membrane in living cells using TIRF microscopy ( Schwarzenbacher et al 2008 ; Lanzerstorfer et al 2014 ; Schütz et al 2017 ; Motsch et al 2019 ). In a recent study, we have demonstrated that this approach is quite robust for studying the activation level of FGFR3 when measuring the interaction with the downstream adaptor protein GRB2 ( Hartl et al 2023 ). Here, we coexpressed each of the different FGFR3 variants (labeled with mGFP) with GRB2 (labeled with mScarlet).…”

Section: Resultsmentioning

confidence: 99%

“…Figure 5b shows the approximate location of the analyzed variants on the protein. The 2 canonical PAE-mutations c.1118A > G (p.Y373C) and c.1138G > A (p.G380R) were previously characterized with the same technique ( Hartl et al 2023 ).…”

Section: Resultsmentioning

confidence: 99%

“…The data are shown as normalized mean values with the respective standard error of the mean of a ratio as described in Supplementary Material onlineand supplementary table S8, Supplementary Material online. The WT, p.Y373C, and p.G380R datasets (nonactivated and FGF1-activated) were previously published in Hartl et al (2023) . The mean of the nonactivated WT (black line) is shown with its confidence interval (gray area).…”

Section: Resultsmentioning

confidence: 99%

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Exploring FGFR3 Mutations in the Male Germline: Implications for Clonal Germline Expansions and Paternal Age-Related Dysplasias

Moura,

Hartl,

Brumovska

et al. 2024

Genome Biology and Evolution

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Delayed fatherhood results in a higher risk of inheriting 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 used digital polymerase chain reaction to assess the frequency and spatial distribution of 10 different FGFR3 missense substitutions in the sexually mature male germline. Our functional assessment of the receptor signaling of the variants with biophysical methods showed that 9 of these variants resulted in a higher activation of the receptor´s downstream signaling, resulting in 2 different expansion behaviors. Variants that form larger subclonal expansions in a dissected postmortem testis also showed a positive correlation of the substitution frequency with the sperm donor's age, and a high and ligand-independent FGFR3 activation. In contrast, variants that measured high FGFR3 signaling and elevated substitution frequencies independent of the donor's age did not result in measurable subclonal expansions in the testis. This suggests that promiscuous signal activation might also result in an accumulation of mutations before the sexual maturation of the male gonad with clones staying relatively constant in size throughout time. Collectively, these results provide novel insights into our understanding of the mutagenesis of driver mutations and their resulting mosaicism in the male germline with important consequences for the transmission and recurrence of associated disorders.

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Section: Discussionmentioning

confidence: 76%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 3 more Smart Citations

Exploring FGFR3 Mutations in the Male Germline: Implications for Clonal Germline Expansions and Paternal Age-Related Dysplasias

Moura,

Hartl,

Brumovska

et al. 2024

Genome Biology and Evolution

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Exploring the Micro-Mosaic Landscape of FGFR3 Mutations in the Ageing Male Germline and Their Potential Implications in Meiotic Differentiation

Striedner,

Arbeithuber,

Moura

et al. 2024

Genes

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Advanced paternal age increases the risk of transmitting de novo germline mutations, particularly missense mutations activating the receptor tyrosine kinase (RTK) signalling pathway, as exemplified by the FGFR3 mutation, which is linked to achondroplasia (ACH). This risk is attributed to the expansion of spermatogonial stem cells carrying the mutation, forming sub-clonal clusters in the ageing testis, thereby increasing the frequency of mutant sperm and the number of affected offspring from older fathers. While prior studies proposed a correlation between sub-clonal cluster expansion in the testis and elevated mutant sperm production in older donors, limited data exist on the universality of this phenomenon. Our study addresses this gap by examining the testis-expansion patterns, as well as the increases in mutations in sperm for two FGFR3 variants—c.1138G>A (p.G380R) and c.1948A>G (p.K650E)—which are associated with ACH or thanatophoric dysplasia (TDII), respectively. Unlike the ACH mutation, which showed sub-clonal expansion events in an aged testis and a significant increase in mutant sperm with the donor’s age, as also reported in other studies, the TDII mutation showed focal mutation pockets in the testis but exhibited reduced transmission into sperm and no significant age-related increase. The mechanism behind this divergence remains unclear, suggesting potential pleiotropic effects of aberrant RTK signalling in the male germline, possibly hindering differentiation requiring meiosis. This study provides further insights into the transmission risks of micro-mosaics associated with advanced paternal age in the male germline.

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Measurement of FGFR3 signaling at the cell membrane via total internal reflection fluorescence microscopy to compare the activation of FGFR3 mutants

Cited by 2 publications

References 60 publications

Exploring FGFR3 Mutations in the Male Germline: Implications for Clonal Germline Expansions and Paternal Age-Related Dysplasias

Exploring FGFR3 Mutations in the Male Germline: Implications for Clonal Germline Expansions and Paternal Age-Related Dysplasias

Exploring the Micro-Mosaic Landscape of FGFR3 Mutations in the Ageing Male Germline and Their Potential Implications in Meiotic Differentiation

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