Pavel Krejci

Fibroblast growth factor receptor 3 (FGFR3) is a membrane protein that plays a critical role as a negative regulator in the growth and development of the skeletal system. Mutations in FGFR3 cause a range of skeletal dysplasias. The G380R mutation in FGFR3 causes achondroplasia (ACH), the most common form of human short‐limbed dwarfism, whereas the A391E mutation in FGFR3 causes Crouzon syndrome, a craniosynostosis characterized by premature fusion of sutures between the cranial bones, with acanthosis nigricans. Both mutations are considered “gain‐of‐function” and occur close to each other within the simple FGFR3 helix that spans the membrane. Yet, the two pathological phenotypes of ACH and Crouzon syndrome are distinctly different. Here, we show that FGFR3 engages in biased signaling. We further show that the G380R and A391E mutations do not simply amplify signaling but differentially reshape the FGFR3 signaling patterns. The data provide an explanation of the distinctly different phenotypes due to the two mutations and lead us to propose that the mutations are “change‐of‐function.”

Background: Fibroblast growth factor 2 (FGF2) is a canonical member of the fibroblast growth factor family with essential roles in embryonic development, tissue regeneration, and stem cell biology, where it supports pluripotency and high proliferative capacity. Consequently, FGF2 is widely used in experimental models of regeneration and stem cell maintenance; however, its rapid thermal degradation at physiological temperatures limits its practical utility and therapeutic potential. Despite the availability of stabilized variants, developing FGF2 analogs that combine exceptional thermodynamic resilience with favorable pharmacokinetic properties remains a critical challenge for stem cell research and regenerative medicine. Results: Using a combination of consensus sequence analysis and structure-guided protein engineering, we generated novel FGF2 variants with unprecedented thermodynamic stability and optimized extracellular matrix interactions. To enhance bioavailability, we introduced mutations that reduced affinity for heparan sulfate proteoglycans, thereby limiting sequestration within the extracellular matrix. The most stable variant exhibited a denaturation temperature increase of more than 27 °C relative to wild-type FGF2 while fully retaining mitogenic and migratory activity after 5 days of incubation at 70 °C. This exceptional thermal stability was accompanied by markedly increased resistance to proteolytic degradation. In functional cellular models, including adipocyte differentiation and stem cell culture, an optimized long-acting variants demonstrated superior metabolic efficacy and sustained signaling, enabling a reduction in dosing frequency from daily administration to once every three days. Conclusions: We report the development of highly stable, highly bioactive FGF2 variants that retain full receptor specificity and binding affinity even under extreme conditions. By overcoming the intrinsic instability of the wild-type protein, these engineered FGF2s may enable future stem cell expansion and regenerative therapy applications.

Osteogenesis imperfecta, a genetically heterogeneous disorder, is characterized by brittle bones with recurrent fractures as well as short stature. A recent study investigating cellular signaling in growth plate chondrocytes identified an increased expression of FGF receptors and elevated FGF signaling. We inhibited FGF receptor activation with the small molecule infigratinib in growing Aga2/+ mice and then characterized linear growth, cartilage growth plate histology, and bone morphometrics. Infigratinib treatment resulted in increased female femur length, increased growth plate lengths in both sexes, specifically in the proliferative zone, and reduced phospho-ERK1/2 as well as SOX9 expression in lower hypertrophic chondrocytes. Infigratinib had no impact on cortical bone but reduced male trabecular bone parameters in Aga2/+ mice. Three point bending biomechanical tests showed that Aga2/+-treated males had increased bone strength for some parameters, but reduced bone strength in treated WT males, correlating with the negative impact on trabecular bone parameters. WT and Aga2-treated male mice also showed increased serum testosterone levels. Together, these data establish FGFR as a therapeutic target for short stature in osteogenesis imperfecta and identify potential negative effects of pan-FGFR inhibition, particularly in the male skeleton.

Fibroblast growth factor 1 (FGF1), a well-characterized member of the FGF family, effectively lowers blood glucose levels in animal models of type 2 diabetes by stimulating glucose uptake. However, its significant mitogenic potential poses a major challenge for clinical application. Here, we present engineered variants of FGF1 designed to dissociate its potent glucose-lowering effects from its undesired proliferative activity, aiming for a future therapeutic agent for type 2 diabetes. Through a series of rational mutations focused on modulating receptor binding and heparan interactions, coupled with enhanced thermodynamic stability, we developed two lead FGF1 variants. Comprehensive in vitro studies confirmed that these variants exhibit significantly reduced mitogenic potential across various cell types compared to wild-type FGF1. Specifically, one variant showed profound loss of proliferation due to disrupted FGFR binding, while the other displayed attenuated mitogenicity linked to decreased heparin affinity. Critically, both fully maintained potent glucose-lowering properties in db/db mice without inducing hypoglycemia or changes in body weight. Furthermore, these engineered proteins demonstrate superior thermodynamic stability and markedly improved pharmacokinetic profile, critical attributes for drug development. Our findings highlight a successful strategy to uncouple the therapeutic benefits of FGF1 from its mitogenic side effects, offering promising, stable, and safe protein-based drug candidates for type 2 diabetes treatment.

Cell communication systems based on polypeptide ligands use transmembrane receptors to transmit signals across the plasma membrane. In their biogenesis, receptors depend on the endoplasmic reticulum (ER)-Golgi system for folding, maturation, transport and localization to the cell surface. ER stress, caused by protein overproduction and misfolding, is a well-known pathology in neurodegeneration, cancer and numerous other diseases. How ER stress affects cell communication via transmembrane receptors is largely unknown. In disease models of multiple myeloma, chronic lymphocytic leukemia and osteogenesis imperfecta, we show that ER stress leads to loss of the mature transmembrane receptors FGFR3, ROR1, FGFR1, LRP6, FZD5 and PTH1R at the cell surface, resulting in impaired downstream signaling. This is caused by downregulation of receptor production and increased intracellular retention of immature receptor forms. Reduction of ER stress by treatment of cells with the chemical chaperone tauroursodeoxycholic acid or by expression of the chaperone protein BiP resulted in restoration of receptor maturation and signaling. We show a previously unappreciated pathological effect of ER stress; impaired cellular communication due to altered receptor processing. Our findings have implications for disease mechanisms related to ER stress and are particularly important when receptor-based pharmacological approaches are used for treatment.

Primary cilium projects from cells to provide a communication platform with neighboring cells and the surrounding environment. This is ensured by the selective entry of membrane receptors and signaling molecules, producing fine-tuned and effective responses to the extracellular cues. In this study, we focused on one family of signaling molecules, the fibroblast growth factor receptors (FGFRs), their residence within cilia, and its role in FGFR signaling. We show that FGFR1 and FGFR2, but not FGFR3 and FGFR4, localize to primary cilia of the developing mouse tissues and in vitro cells. For FGFR2, we demonstrate that the ciliary residence is necessary for its signaling and expression of target morphogenic genes. We also show that the pathogenic FGFR2 variants have minimal cilium presence, which can be rescued for the p.P253R variant associated with the Apert syndrome by using the RLY-4008 kinase inhibitor. Finally, we determine the molecular regulators of FGFR2 trafficking to cilia, including IFT144, BBS1, and the conserved T429V430 motif within FGFR2.

Fibroblast growth factors (FGFs) control organ morphogenesis during development as well as tissue homeostasis and repair in the adult organism. Despite their importance, many mechanisms that regulate FGF function are still poorly understood. Interestingly, the thermodynamic stability of 22 mammalian FGFs varies widely, with some FGFs remaining stable at body temperature for more than 24 h, while others lose their activity within minutes. How thermodynamic stability contributes to the function of FGFs during development remains unknown. Here we show that FGF10, an important limb and lung morphogen, exists as an intrinsically unstable protein that is prone to unfolding and is rapidly inactivated at 37 °C. Using rationally driven directed mutagenesis, we have developed several highly stable (STAB) FGF10 variants with a melting temperature of over 19 °C more than that of wildtype FGF10. In cellular assays in vitro, the FGF10-STABs did not differ from wildtype FGF10 in terms of binding to FGF receptors, activation of downstream FGF receptor signaling in cells, and induction of gene expression. In mouse embryonal lung explants, FGF10-STABs, but not wildtype FGF10, suppressed branching, resulting in increased alveolarization and expansion of epithelial tissue. Similarly, FGF10-STAB1, but not FGF10 wildtype, inhibited the growth of mouse embryonic tibias and markedly altered limb morphogenesis when implanted into chicken limb buds, collectively demonstrating that thermal instability should be considered an important regulator of FGF function that prevents ectopic signaling. Furthermore, we show enhanced differentiation of human iPSC-derived lung organoids and improved regeneration in ex vivo lung injury models mediated by FGF10-STABs, suggesting an application in cell therapy.

Most of our knowledge of protein structure and function originates from experiments performed with purified proteins resuspended in dilute, buffered solutions. However, most proteins function in crowded intracellular environments with complex compositions. Significant efforts have been made to develop tools to study proteins in their native cellular settings. Among these tools, in-cell NMR spectroscopy has been the sole technique for characterizing proteins in the intracellular space of living cells at atomic resolution and physiological temperature. Nevertheless, due to technological constraints, in-cell NMR studies have been limited to asynchronous single-cell suspensions, precluding obtaining information on protein behavior in different cellular states. In this study, we present a methodology that allows for obtaining an atomically resolved NMR readout of protein structure and interactions in living human cells synchronized in specific cell cycle phases and within 3D models of human tissue. The described approach opens avenues for investigating how protein structure or drug recognition responds to cell-cell communication or changes in intracellular space composition during transitions among cell cycle phases.