Dissecting biological activities of fibroblast growth factor receptors by the coiled-coil-mediated oligomerization of FGF1

Porębska, Natalia; Pozniak, Marta; Krzyscik, Mateusz Adam; Knapik, Agata; Czyrek, Aleksandra; Kucińska, Marika; Jastrzębski, Kamil; Zakrzewska, Małgorzata; Otlewski, Jacek; Opaliński, Łukasz

doi:10.1016/j.ijbiomac.2021.03.094

Cited by 12 publications

(20 citation statements)

References 42 publications

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“…While all FGF1 oligomers were capable of FGFR1 binding and activation, trimeric and tetrameric FGF1 displayed largely increased affinity for FGFR1 in comparison to the monomeric ligand. We have previously observed a similar phenomenon for tetravalent anti-FGFR1 antibody and coiled-coil-triggered FGF1 oligomers and in both cases the elevated affinity for FGFR1 was due to considerably decreased dissociation rates [ 25 , 46 ]. Importantly, improved FGFR1 binding was accompanied by enhanced cellular uptake of oligomeric FGF1, indicating that SA-FGF1 multivalent ligands, by affecting the spatial distribution of FGFR1, control its cellular trafficking and may constitute effective targeting molecules for selective drug delivery.…”

Section: Discussionmentioning

confidence: 61%

Modular self-assembly system for development of oligomeric, highly internalizing and potent cytotoxic conjugates targeting fibroblast growth factor receptors

et al. 2021

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Background Overexpression of FGFR1 is observed in numerous tumors and therefore this receptor constitutes an attractive molecular target for selective cancer treatment with cytotoxic conjugates. The success of cancer therapy with cytotoxic conjugates largely relies on the precise recognition of a cancer-specific marker by a targeting molecule within the conjugate and its subsequent cellular internalization by receptor mediated endocytosis. We have recently demonstrated that efficiency and mechanism of FGFR1 internalization are governed by spatial distribution of the receptor in the plasma membrane, where clustering of FGFR1 into larger oligomers stimulated fast and highly efficient uptake of the receptor by simultaneous engagement of multiple endocytic routes. Based on these findings we aimed to develop a modular, self-assembly system for generation of oligomeric cytotoxic conjugates, capable of FGFR1 clustering, for targeting FGFR1-overproducing cancer cells. Methods Engineered FGF1 was used as FGFR1-recognition molecule and tailored for enhanced stability and site-specific attachment of the cytotoxic drug. Modified streptavidin, allowing for controlled oligomerization of FGF1 variant was used for self-assembly of well-defined FGF1 oligomers of different valency and oligomeric cytotoxic conjugate. Protein biochemistry methods were applied to obtain highly pure FGF1 oligomers and the oligomeric cytotoxic conjugate. Diverse biophysical, biochemical and cell biology tests were used to evaluate FGFR1 binding, internalization and the cytotoxicity of obtained oligomers. Results Developed multivalent FGF1 complexes are characterized by well-defined architecture, enhanced FGFR1 binding and improved cellular uptake. This successful strategy was applied to construct tetrameric cytotoxic conjugate targeting FGFR1-producing cancer cells. We have shown that enhanced affinity for the receptor and improved internalization result in a superior cytotoxicity of the tetrameric conjugate compared to the monomeric one. Conclusions Our data implicate that oligomerization of the targeting molecules constitutes an attractive strategy for improvement of the cytotoxicity of conjugates recognizing cancer-specific biomarkers. Importantly, the presented approach can be easily adapted for other tumor markers.

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Section: Discussionmentioning

confidence: 61%

Modular self-assembly system for development of oligomeric, highly internalizing and potent cytotoxic conjugates targeting fibroblast growth factor receptors

et al. 2021

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“…Regulation of RTK distribution in the plasma membrane is one of strategies for fine-tuning RTK-dependent signaling [10]. We have previously reported that by using engineered oligomeric FGFR1 ligands it is possible to control activity, endocytosis and cellular level of the receptor [21,22]. Importantly, the architecture of the oligomeric ligands (binding site on the receptor, affinity, oligomeric state, oligomerization scaffold used) played an important role in determining the outcome of FGFR1 modulation [21,22].…”

Section: Discussionmentioning

confidence: 99%

“…We have previously reported that by using engineered oligomeric FGFR1 ligands it is possible to control activity, endocytosis and cellular level of the receptor [21,22]. Importantly, the architecture of the oligomeric ligands (binding site on the receptor, affinity, oligomeric state, oligomerization scaffold used) played an important role in determining the outcome of FGFR1 modulation [21,22]. Therefore, we have sought to develop novel strategies to generate multivalent FGFR1 ligands.…”

Section: Discussionmentioning

confidence: 99%

“…So far, we have been able to obtain FGFR1 ligands having up to five receptor binding sites [22]. In this report, we present an approach that allows the efficient assembly of FGFR1-targeting oligomers with custom-selected, higher number of receptor binding sites.…”

Section: Discussionmentioning

confidence: 99%

“…Importantly, FGFR1 clustering alters the mechanisms of receptor internalization by activating clathrin-independent, dynamin-dependent endocytic routes [21]. Thirdly, tetravalent and pentavalent FGF1 oligomers alter FGFR1 activity by promoting mitogenic signaling and inhibiting cell metabolic activity [22].…”

Section: Introductionmentioning

confidence: 99%

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FGF1 Fusions with the Fc Fragment of IgG1 for the Assembly of GFPpolygons-Mediated Multivalent Complexes Recognizing FGFRs

et al. 2021

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FGFRs are cell surface receptors that, when activated by specific FGFs ligands, transmit signals through the plasma membrane, regulating key cellular processes such as differentiation, division, motility, metabolism and death. We have recently shown that the modulation of the spatial distribution of FGFR1 at the cell surface constitutes an additional mechanism for fine-tuning cellular signaling. Depending on the multivalent, engineered ligand used, the clustering of FGFR1 into diverse supramolecular complexes enhances the efficiency and modifies the mechanism of receptor endocytosis, alters FGFR1 lifetime and modifies receptor signaling, ultimately determining cell fate. Here, we present a novel approach to generate multivalent FGFR1 ligands. We functionalized FGF1 for controlled oligomerization by developing N- and C-terminal fusions of FGF1 with the Fc fragment of human IgG1 (FGF1-Fc and Fc-FGF1). As oligomerization scaffolds, we employed GFPpolygons, engineered GFP variants capable of well-ordered multivalent display, fused to protein G to ensure binding of Fc fragment. The presented strategy allows efficient assembly of oligomeric FGFR1 ligands with up to twelve receptor binding sites. We show that multivalent FGFR1 ligands are biologically active and trigger receptor clustering on the cell surface. Importantly, the approach described in this study can be easily adapted to oligomerize alternative growth factors to control the activity of other cell surface receptors.

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Receptor clustering by a precise set of extracellular galectins initiates FGFR signaling

Zukowska

Gedaj

Porębska

et al. 2023

Cell. Mol. Life Sci.

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FGF/FGFR signaling is critical for the development and homeostasis of the human body and imbalanced FGF/FGFR contributes to the progression of severe diseases, including cancers. FGFRs are N-glycosylated, but the role of these modifications is largely unknown. Galectins are extracellular carbohydrate-binding proteins implicated in a plethora of processes in heathy and malignant cells. Here, we identified a precise set of galectins (galectin-1, -3, -7, and -8) that directly interact with N-glycans of FGFRs. We demonstrated that galectins bind N-glycan chains of the membrane-proximal D3 domain of FGFR1 and trigger differential clustering of FGFR1, resulting in activation of the receptor and initiation of downstream signaling cascades. Using engineered galectins with controlled valency, we provide evidence that N-glycosylation-dependent clustering of FGFR1 constitutes a mechanism for FGFR1 stimulation by galectins. We revealed that the consequences of galectin/FGFR signaling for cell physiology are markedly different from the effects induced by canonical FGF/FGFR units, with galectin/FGFR signaling affecting cell viability and metabolic activity. Furthermore, we showed that galectins are capable of activating an FGFR pool inaccessible for FGF1, enhancing the amplitude of transduced signals. Summarizing, our data identify a novel mechanism of FGFR activation, in which the information stored in the N-glycans of FGFRs provides previously unanticipated information about FGFRs’ spatial distribution, which is differentially deciphered by distinct multivalent galectins, affecting signal transmission and cell fate.

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Dissecting biological activities of fibroblast growth factor receptors by the coiled-coil-mediated oligomerization of FGF1

Cited by 12 publications

References 42 publications

Modular self-assembly system for development of oligomeric, highly internalizing and potent cytotoxic conjugates targeting fibroblast growth factor receptors

Modular self-assembly system for development of oligomeric, highly internalizing and potent cytotoxic conjugates targeting fibroblast growth factor receptors

FGF1 Fusions with the Fc Fragment of IgG1 for the Assembly of GFPpolygons-Mediated Multivalent Complexes Recognizing FGFRs

Receptor clustering by a precise set of extracellular galectins initiates FGFR signaling

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