Ligand bias underlies differential signaling of multiple FGFs via FGFR1

Joint Public Review

In this manuscript, Karl et al. explore mechanisms underlying the activation of the receptor tyrosine kinase FGFR1 and stimulation of intracellular signaling pathways in response to FGF4, FGF8, or FGF9 binding to the extracellular domain of FGFR1. Quantitative binding experiments presented in the manuscript demonstrate that FGF4, FGF8, and FGF9 exhibit distinct binding affinities towards FGFRs. It is also proposed that FGF8 exhibits "biased ligand" characteristics that is manifested via binding and activation FGFR1 mediated by "structural differences in the FGF- FGFR1 dimers, which impact the interactions of the FGFR1 trans membrane helices, leading to differential recruitment and activation of the downstream signaling adapter FRS2".

In the absence of any structural experimental data of different forms of FGFR dimers stimulated by FGF ligands the model presents in the manuscript is speculative and misleading.

Author Response

The following is the authors’ response to the original reviews.

In response to the eLife assessment that “the analysis of the data is inadequate”, we strongly disagree and we to point out that in fact we follow the latest IUPHAR community guidelines on bias identification and quantification (Kolb et al, 2022). These protocols are not yet being used in the RTK and FGF fields, and thus the reviewer is not familiar with them, or with the concept of ligand bias. Our responses to the technical comments start at the bottom of page 7 of this document.

We have edited the paper by adding a scaling step-by-step protocol in the Supplementary Data. We have also expanded the Discussion to help readers understand what is measured and how it is very novel. We have also changed the title of the manuscript. The edits in the Manuscript are marked in yellow. Our response to the reviewer is given below.

Question/comment: 1. Previous studies have demonstrated that the variability of signal transduction stimulated by different FGF family members originates from their preferential activation of different members of the FGFR family (Ornitz et al., 1996). For example, it was previously shown that members of the FGF8 subfamily preferentially activate FGFR3c, whereas members of the FGF4 subfamily activate FGFR1c more potently than other FGFs. Moreover, it was shown that FGF18, a member of the FGF8 subfamily, preferentially binds to and activates the FGFR3c isoform. Indeed, this can be seen in the data shown in Figure 3 in this manuscript, where maximum levels of FGFR1 pY653/4 and pFRS2 are reached at different concentrations when stimulated with increasing concentrations of each ligand in HEK293T cells.

The reviewer is correct that there are differences in the signaling of the different FGFRs, however these differences are not relevant for this work. This paper is only about FGFR1c, as this is the only FGF-receptor which is expressed in the mesenchyme of the developing limb bud (early limb bud stage, before the onset of mesenchymal condensations) and encounters different FGF ligands. In the article, we analyze the mechanism by which one FGFR recognizers and responds to three different FGFs.

The reviewer also correctly points out that differences in our work “can be seen in the data shown in Figure 3 in this manuscript, where maximum levels of FGFR1 pY653/4 and pFRS2 are reached at different concentrations when stimulated with increasing concentrations of each ligand in HEK293T cells”. This is correct, but this is a statement about the potencies of the ligands, which is just one of three characteristics we explore here, namely potencies, efficacies, and bias. To determine if ligand bias exists or not, we need to compare two ligands and two responses (such as growth arrest and ECM degradation, or pY653/4 and pFRS2 phosphorylation). Ours is the first report of ligand bias in FGFR1 signaling, and the presence of bias goes far beyond simply differences in potencies (Kolb et al, 2022). Ligand bias in FGFR1 has never been demonstrated before. In part, this is because there have been no cell lines that give us the opportunity to compare two functional responses to FGF stimulus, via just one endogenously expressed FGFR variant. Notice that the paper that the reviewer is citing, (Ornitz et al., 1996), compares only 1 (one) type of response, when induced by different ligands, i.e. proliferation, and thus cannot answer the question if ligand bias exists or not. We have edited the Discussion to emphasize this fact. We have also changed the title.

Two studies meant to characterize FGF binding to the FGFRs (Ornitz et al., 1996; Zhang et al., 2006) have defined the main rules of the FGF-FGFR interaction, such as exclusivity of the FGF3 subfamily (FGF3, FGF7, FGF10) for the ‘b’ variants of the FGFR1 and FGFR2. These studies however do not measure ligand binding. These studies were carried-out in BAF/3 cells, where the transfected FGFRs are treated with exogenous FGFs, to cause cell proliferation. As such, the studies have several limitations. In BAF/3 cells, the cell proliferation is used as a surrogate for FGF binding on FGFR. The FGFRs activate cell proliferation via RAS-ERK MAP kinase pathway. However, many other pathways of downstream signaling are initiated by FGFRs, regulating cell differentiation, migration, metabolism and apoptosis, in biological contexts. Using single cellular response (cell proliferation) as a surrogate for FGF binding to their receptors will favor FGF ligands causing cell proliferation. FGFs which have preference for other responses will incorrectly appear weakly binding and weakly activating in BAF/3 cells. Further, an FGF ligand binding with high affinity to the receptor but inducing a lower proliferative response will be recognized as a less ‘preferential’ for the particular receptor in the BAF/3 assay. Second, the significant diversity of signaling of 18 FGFs through seven FGFR variants in mammalian development suggests that many previously unappreciated nodules of FGF-FGFR signaling exist, including the recently discovered FGF signaling towards primary cilia, or interaction with insulin receptor system (Kunova Bosakova et al., 2019; Neugebauer et al., 2009; Nies et al., 2022). This diversity is not reflected in BAF/3 assay, which respond to FGFs with only one phenotype. This is why we have used the RCS cells in the manuscript. In RCS cells, at least two qualitatively different cell responses can be induced by the FGF signaling, making the cell model ideal for elucidating biased signaling.

The so called ‘binding preferences’ based on the Ornitz articles are not binding measurements and should not be used universally to describe the FGF interactions with FGFRs, because we do not know what the term really means, nor what is it based on; the molecular basis of the FGFR signaling BAF/3 is poorly characterized. In our article, we model the processes occurring in every developing mammalian limb, where three FGF ligands (FGF4, FGF8, FGF9), released by the ectoderm at the surface of the limb bud, signal to the underlying mesenchymal cell expressing just one FGF-receptor, the FGFR1c (Mariani and Martin, 2003; Tabin and Wolpert, 2007). Unlike the BAF/3 cells engineered to ectopically express one FGFR and treated by recombinant FGFs in the lab, all three FGFs are recognized by cells expressing FGFR1c, and each of the three FGFs delivers unique morphogenetic information. The mechanisms underlying differential signaling of multiple FGFs via one FGFR are poorly defined, as the term ‘preferential signaling’ does not provide mechanistic explanation. Our article is a step towards understanding the complex processes of FGF ligand recognition and response. In our article, we evaluate the potency, the efficacy, the FGFinduced FGFR1c oligomerization and downregulation, and conformation of the active FGFR1c dimers in response to FGF4, FGF8 and FGF9. We show that FGF4, FGF8, and FGF9 are biased ligands, and that bias can explain differences in FGF4, FGF8 and FGF9-mediated cellular responses in development.

References

Kolb P, Kenakin T, Alexander SPH, Bermudez M, et al. Community guidelines for GPCR ligand bias: IUPHAR review 32. Br J Pharmacol. 2022;179, 3651-3674.

Kunova Bosakova M, Nita A, Gregor T, Varecha M, et al. Fibroblast growth factor receptor influences primary cilium length through an interaction with intestinal cell kinase. Proc Natl Acad Sci U S A. 2019;116(10):4316-4325.

Mariani FV, Martin GR. Deciphering skeletal patterning: clues from the limb. Nature. 2003;423(6937):319-25.

Nies VJM, Struik D, Liu S, Liu W, et al. Autocrine FGF1 signaling promotes glucose uptake in adipocytes. Proc Natl Acad Sci U S A. 2022;119(40):e2122382119.

Neugebauer JM, Amack JD, Peterson AG, Bisgrove BW, Yost HJ. FGF signalling during embryo development regulates cilia length in diverse epithelia. Nature. 2009;458(7238):651-4.

Ornitz DM, Xu J, Colvin JS, McEwen DG, et al. Receptor specificity of the fibroblast growth factor family. J Biol Chem. 1996;271(25):15292-7.

Tabin C, Wolpert L. Rethinking the proximodistal axis of the vertebrate limb in the molecular era. Genes Dev. 2007;21(12):1433-42.

Zhang X, Ibrahimi OA, Olsen SK, Umemori H, Mohammadi M, Ornitz DM. Receptor specificity of the fibroblast growth factor family. The complete mammalian FGF family. J Biol Chem. 2006;281(23):15694-700.

Question/comment: In order to be sure that the 'biased agonist' described in this manuscript for FGF8 binding is not caused by binding preference towards different FGFR members, the authors should present data comparing cell signaling via FGFR3c stimulated by FGF4, FGF8, and FGF9.

Here, we study signaling by FGFR1, which is the only receptor that is expressed in the mesenchyme of the developing limb bud. FGFR3 is not expressed there, and thus we do not study FGFR3 in this paper. FGFR3 is important regulator of skeletal development, but is not involved in the early stages like FGFR1. When the bones are formed, FGFR3 regulates chondrocyte proliferation and differentiation in the growth plate cartilage (Colvin et al., 1996). In fact, we are currently performing experiments with FGFR3 and multiple FGF ligands, and we see that it also engages in biased signaling. However, these FGFR3 studies have no relevance to the current work and will be published separately.

The so called ‘binding preferences towards different FGFR members’, based on the Ornitz articles (Ornitz et al., 1996; Zhang et al., 2006) provides no mechanistic explanation about differential FGF signaling via the activation of a single FGFR. Our article is a step forward towards the mechanism, by demonstration, for the first time, that ‘ligand bias’ may explain differential signaling by FGF4, FGF8 and FGF9 via FGFR1c.

References

Colvin JS, Bohne BA, Harding GW, McEwen DG, Ornitz DM. Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3. Nat Genet. 1996;12(4):390-7.

Ornitz DM, Xu J, Colvin JS, McEwen DG, MacArthur CA, Coulier F, Gao G, Goldfarb M. Receptor specificity of the fibroblast growth factor family. J Biol Chem. 1996;271(25):15292-7.

Zhang X, Ibrahimi OA, Olsen SK, Umemori H, Mohammadi M, Ornitz DM. Receptor specificity of the fibroblast growth factor family. The complete mammalian FGF family. J Biol Chem. 2006;281(23):15694-700.

Question/comment: 2. It is well-established that FGFR signaling by canonical FGF family members including FGF4, FGF8, and FGF9 is dependent on interactions of heparin or heparan sulfate proteoglycans (HSPG) to the ligand the receptors. Differential contributions of heparin to cell signaling mediated by FGF4, FGF8, and FGF9 binding and activation of different FGFRs expressed in RCS cells as this cell express endogenous HSPG molecules. This question should be addressed by comparing cell signaling via FGFRs ectopically expressed in BAF/3 cells (which do not possess endogenous FGFRs and HSPG) stimulated by FGF4, FGF8, and FGF9 in the absence or presence of different heparin concentrations. This approach has been applied many times in the past to explore and establish the role of heparin in control of ligand induced FGFR activation.

The work cannot be done with BAF/3 cells, since the topic of the study is ligand bias so we need to compare at least two measurable responses. In RCS cells, the two functional responses are growth arrest and extracellular matrix degradation. In BAF/3 cells, ligand stimulation leads to one single response: proliferation.

The HSPG and other sulphated proteoglycans work as low affinity FGF co-receptors. They stabilize the FGF secondary structure, present the FGFs to the FGFRs, and participate in FGFFGFR interactions (Yayon et al., 1991; Schlessinger et al., 2000; Zakrzewska et al., 2009). In the FGF field, the FGF-FGFR interaction is commonly supported by addition of exogenous heparin, which is highly sulphated glycosaminoglycan capable of full substitution of the cell-bound HSPGs in their function as low affinity FGF co-receptors.

Most cells produce proteoglycans, including BAF/3 cells. The analysis of expression of FGFR overexpressed in BAF/3 cells demonstrated that FGFR1, FGFR2 and FGFR3 migrate as proteins of approximately 130-150 kDa (Ornitz et al., 1996; Fig. 1A), which implies extensive glycosylation in Golgi. For instance, the full-length amino acid sequence for human FGFR3 is 806 residues, which on acrylamide gel migrates as a band of approximately 85 kDa; heavier FGFR3 variants are Golgi-glycosylated proteins. The treatment with de-glycosylation enzymes reduces the molecular weight to the one expected from the amino acid sequence.

To carry-out the BAF/3 experiment with FGF4, FGF8, and FGF9 in the absence or presence of different heparin concentrations, as the referee suggests, makes no sense. In BAF/3 cells, all FGF stimulations were done in the presence of 2 g/ml heparin (Ornitz et al., 1996; Zhang et al., 2006), because without heparin there would be no signaling. Even if the BAF/3 cells produce ample HSPGs, the heparin would still have to be used, because without it many of the FGFs would likely cause no response, regardless of the FGFR variant expressed. We and other have demonstrated, that most of the FGFs require stabilization by heparin to elicit signaling in cells expressing abundant amounts of HSPG (Buchtova et al., 2015; Chen et al., 2012).

Why should we compare the FGF signaling in BAF/3 transfected with FGFR1, with the RCS cells which express endogenous FGFR1? In RCS cells, several cellular phenotypes caused by FGF signaling can be easily detected and quantified, in comparison with BAF/3 cells, which only respond to the FGF signaling by proliferation. No bias in signaling can be established in cells with display only single type of response. The RCS cells used in our paper represent one of the most tractable cellular models of FGFR signaling. There are more than 40 articles exploring the mechanisms of FGF-FGFR signaling in RCS cells, including mechanisms of FGF signal transduction, FGF regulation of cell cycle, cell proliferation, differentiation, premature senescence, loss of extracellular matrix, interaction of FGF signaling with WNT, cytokine and natriuretic peptide signaling, and others (Raucci et al., 2004; Priore et al., 2006; Kamemura et al., 2017; Kolupaeva et al., 2013; Krejci et al., 2005; Krejci et al., 2007; Krejci et al., 2010; Dailey et al., 2003; Rozenblatt-Rosen et al., 2002; Fafilek et al., 2008). In addition, the three treatments to inhibit pathological FGFR signaling which are now in human trials (RBM007, meclozine) or FDAapproved (vosoritide), were initially developed in RCS cells, benefiting from the well characterized molecular mechanisms of FGF signaling (Krejci et al., 2005; Wendt et al., 2015; Kimura et al., 2021; Matsushita et al., 2013). In comparison with RCS cells, very little is known about the mechanisms of the FGF signaling in BAF/3 cells, as the BAF/3 proliferation assay is used mostly to evaluate FGFR agonists and antagonists (Yamada et al., 2020; Kamatkar et al., 2019; Motomura et al., 2008). We have edited this information to the revised Discussion.

References

Buchtova M, Oralova V, Aklian A, Masek J, et al. Fibroblast growth factor and canonical WNT/βcatenin signaling cooperate in suppression of chondrocyte differentiation in experimental models of FGFR signaling in cartilage. Biochim Biophys Acta. 2015 May;1852(5):839-50.

Buchtova M, Chaloupkova R, Zakrzewska M, Vesela I, et al. Instability restricts signaling of multiple fibroblast growth factors. Cell Mol Life Sci. 2015 Jun;72(12):2445-59.

Chen G, Gulbranson DR, Yu P, Hou Z, Thomson JA. Thermal stability of fibroblast growth factor protein is a determinant factor in regulating self-renewal, differentiation, and reprogramming in human pluripotent stem cells. Stem Cells. 2012 Apr;30(4):623-30.

Fafilek B, Balek L, Bosakova MK, Varecha M, et al. The inositol phosphatase SHIP2 enables sustained ERK activation downstream of FGF receptors by recruiting Src kinases. Sci Signal. 2018 Sep 18;11(548):eaap8608.

Kamemura N, Murakami S, Komatsu H, Sawanoi M, et al. Biochem Biophys Res Commun. 2017 Jan 29;483(1):82-87.

Kamatkar N, Levy M, Hébert JM. Development of a Monomeric Inhibitory RNA Aptamer Specific for FGFR3 that Acts as an Activator When Dimerized. Mol Ther Nucleic Acids. 2019 Sep 6;17:530-539.

Kimura T, Bosakova M, Nonaka Y, Hruba E, Yasuda K, et al. An RNA aptamer restores defective bone growth in FGFR3-related skeletal dysplasia in mice. Sci Transl Med. 2021 ;13(592):eaba4226.

Kolupaeva V, Daempfling L, Basilico C. The B55α regulatory subunit of protein phosphatase 2A mediates fibroblast growth factor-induced p107 dephosphorylation and growth arrest in chondrocytes. Mol Cell Biol. 2013 Aug;33(15):2865-78.

Krejci P, Masri B, Salazar L, Farrington-Rock C, et al. Bisindolylmaleimide I suppresses fibroblast growth factor-mediated activation of Erk MAP kinase in chondrocytes by preventing Shp2 association with the Frs2 and Gab1 adaptor proteins. J Biol Chem. 2007;282(5):2929-36.

Krejci P, Masri B, Fontaine V, Mekikian PB, et al. Interaction of fibroblast growth factor and C-natriuretic peptide signaling in regulation of chondrocyte proliferation and extracellular matrix homeostasis. J Cell Sci. 2005 Nov 1;118(Pt 21):5089-100.

Krejci P, Prochazkova J, Smutny J, Chlebova K, et al. FGFR3 signaling induces a reversible senescence phenotype in chondrocytes similar to oncogene-induced premature senescence. Bone. 2010;47(1):102-10.

Matsushita M, Kitoh H, Ohkawara B, Mishima K, et al. Meclozine facilitates proliferation and differentiation of chondrocytes by attenuating abnormally activated FGFR3 signaling in achondroplasia. PLoS One. 2013;8(12):e81569.

Motomura K, Hagiwara A, Komi-Kuramochi A, Hanyu Y, et al. An FGF1:FGF2 chimeric growth factor exhibits universal FGF receptor specificity, enhanced stability and augmented activity useful for epithelial proliferation and radioprotection. Biochim Biophys Acta. 2008 Dec;1780(12):1432-40.

Ornitz DM, Xu J, Colvin JS, McEwen DG, MacArthur CA, Coulier F, Gao G, Goldfarb M. Receptor specificity of the fibroblast growth factor family. J Biol Chem. 1996;271(25):15292-7.

Priore R, Dailey L, Basilico C. Downregulation of Akt activity contributes to the growth arrest induced by FGF in chondrocytes. J Cell Physiol. 2006 Jun;207(3):800-8.

Raucci A, Laplantine E, Mansukhani A, Basilico C. Activation of the ERK1/2 and p38 mitogen-activated protein kinase pathways mediates fibroblast growth factor-induced growth arrest of chondrocytes. J Biol Chem. 2004;279(3):1747-56.

Robinson JW, Egbert JR, Davydova J, Schmidt H, et al. Dephosphorylation is the mechanism of fibroblast growth factor inhibition of guanylyl cyclase-B. Cell Signal. 2017;40:222229.

Rozenblatt-Rosen O, Mosonego-Ornan E, Sadot E, Madar-Shapiro L, et al. Induction of chondrocyte growth arrest by FGF: transcriptional and cytoskeletal alterations. J Cell Sci. 2002 Feb 1;115(Pt 3):553-62.

Schlessinger J, Plotnikov AN, Ibrahimi OA, Eliseenkova AV, et al. Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization. Mol Cell. 2000 Sep;6(3):743-50.

Wendt DJ, Dvorak-Ewell M, Bullens S, Lorget F, et al. Neutral endopeptidase-resistant Ctype natriuretic peptide variant represents a new therapeutic approach for treatment of fibroblast growth factor receptor 3-related dwarfism. J Pharmacol Exp Ther. 2015 Apr;353(1):132-49.

Yamada R, Fukumoto R, Noyama C, Fujisawa A, et al. An epidermis-permeable dipeptide is a potential cosmetic ingredient with partial agonist/antagonist activity toward fibroblast growth factor receptors. J Cosmet Dermatol. 2020 Feb;19(2):477-484.

Yayon A, Klagsbrun M, Esko JD, Leder P, Ornitz DM. Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell. 1991 Feb 22;64(4):841-8.

Zakrzewska M, Wiedlocha A, Szlachcic A, Krowarsch D, et al. Increased protein stability of FGF1 can compensate for its reduced affinity for heparin. J Biol Chem. 2009 Sep 11;284(37):25388-403. doi: 10.1074/jbc.M109.001289.

Zhang X, Ibrahimi OA, Olsen SK, Umemori H, Mohammadi M, Ornitz DM. Receptor specificity of the fibroblast growth factor family. The complete mammalian FGF family. J Biol Chem. 2006;281(23):15694-700.

Question/comment: It is impossible to interpret the FGFR binding characteristics and cellular activates of FGF4, FGF8, and FGF9 in the absence of information about the role of heparin in their binding and activation.

We do not measure ligand binding to FGFR1 in this study. We record biological responses when we treat with FGF different ligands, and thus we measure the efficacy and the potency of each ligand to induce a response, and then we compare 2 ligands and 2 responses to determine if bias exists or not. We do not ask questions about the role of heparin, as it is always there no matter if we treat with FGF4, FGF8, or FGF9.

Why it is not possible to interpret our cellular data? In our article, the RCS cells were treated with FGFs in the presence of 1 g/ml heparin, as clearly stated in Methods section. Using heparin at 1 or more μg/ml, to stabilize FGFs and negate the effect of endogenous HSPG, is a standard approach in the FGF field. This includes the two articles, which the whole field have used for more than 20 years as a basic reference for FGF-FGFR interactions (Ornitz et al., 1996; Zhang et al., 2006). In these studies, 2 μg/ml of heparin along with FGFs was used to treat BAF/3 cells; no experiments were conducted without heparin, as is does not make sense. Most likely, without heparin the obtained FGF-FGFR ‘preferences’ would, in fact, be the differences in FGF thermal stability, as we clearly demonstrate in our previous study (Buchtova et al., 2015). The latter article gives a detailed information about the role of heparin in the signaling of multiple FGFs in RCS cells.

References

Buchtova M, Chaloupkova R, Zakrzewska M, Vesela I, Cela P, Barathova J, Gudernova I, Zajickova R, Trantirek L, Martin J, Kostas M, Otlewski J, Damborsky J, Kozubik A, Wiedlocha A, Krejci P. Instability restricts signaling of multiple fibroblast growth factors. Cell Mol Life Sci. 2015 Jun;72(12):2445-59.

Ornitz DM, Xu J, Colvin JS, McEwen DG, MacArthur CA, Coulier F, Gao G, Goldfarb M. Receptor specificity of the fibroblast growth factor family. J Biol Chem. 1996;271(25):15292-7.
Zhang X, Ibrahimi OA, Olsen SK, Umemori H, Mohammadi M, Ornitz DM. Receptor specificity of the fibroblast growth factor family. The complete mammalian FGF family. J Biol Chem. 2006;281(23):15694-700.

Technical Comments/Answers

Question/comment: 3. It is not clear how some of the experimental data were analyzed. Blots in Figures 3A and 3B should include controls (total FGFR1 for pY653/4 and total FRS for pFRS2). How are the data shown in Figure 3C normalized? It does look like the level of phosphorylation was all normalized against the strongest signals irrespective of which ligand was used. Each data representing each ligand should be separately normalized.

The reviewer is correct that most often in the RTK literature “each data representing each ligand is separately normalized”. But this approach will eliminate all the information about ligand efficacies and about ligand bias; it will only yield information about the potencies. Here we are not only interested in the potencies, as we are also interested to determine if bias exists or not. As such, we follow scaling protocols that have been established and are currently recommended for ligand bias studies (Kolb et al, 2022).

One way to explain why the scaling that the reviewer is recommending is not correct for this work is to look at equation 2. What the reviewer is suggestion is to set all values of Etop to 1. In this case, the bias coefficient will depend only on the measured potencies, EC50. But this contradicts the very definition of bias, as it is NOT a difference in potencies only. In the literature, differences in potencies are called “quantitative differences”, while ligand bias describes differences which are called “qualitative” or “fundamental” (Kenakin, 2019).

To eliminate confusion, we have added a scaling protocol to the Supplement of the paper.

References

Kolb P, Kenakin T, Alexander SPH, Bermudez M, et al. Community guidelines for GPCR ligand bias: IUPHAR review 32. Br J Pharmacol. 2022;179, 3651-3674.

Kenakin T. Biased Receptor Signaling in Drug Discovery. Pharmacol Rev 2019;71, 267315.

Question/comment: 4. In page 6, authors used the plot shown in Figure 3 for 'FGFR downregulation' to conclude that "the effect of FGF4 on FGFR1 downregulation is smaller when compared to the effects of FGF8 and FGF9. However, it is unclear how the data shown in the plot was normalized - none of the data seem to reach "1.0". Moreover, the plot seems to suggest that FGF4 can strongly downregulate FGFR as it can downregulate FGFR with higher potency.

The Western blots assessing FGFR1 expression are easy to scale, as the value in the absence of ligand is set to 1. The expression decreases as a function of the ligand concentration. We plot FGFR1 downregulation, so we subtract 1 from the scaled FGFR1 band intensities. The total amount of FGFR1 never becomes undetectable (i.e. zero), as the ligand concentration is increased. Thus, a value of 1 in the downregulation curve is never obtained.

We have added a protocol for this scaling in the Supplement.

Question/comment: 5. The structural basis of FGFR1 ligand bias and the different dimeric configurations and interactions between the kinase domain of FGFR1 dimers are not warranted (Figure 6). In the absence of any structural experimental data of different forms of FGFR dimers stimulated by FGF ligands the model presents in the manuscript is speculative and misleading.

This statement about Figure 6 is not fully correct because Figure 6A and B show experimental data. These are FRET experiments which show that the biased ligand, FGF8, induces different FGFR1 transmembrane domain conformation, as compared to FGF4 and FGF9.

The rest of the panels in Figure 6 show modeling using PyRosetta. These are indeed not experimental data, but to the best of our knowledge this is the very first time PyRosetta has been used to predict kinase-kinase interfaces.