Unliganded fibroblast growth factor receptor 1 forms density-independent dimers.
Fibroblast growth factors receptors (FGFRs) are thought to initiate intracellular signaling cascades upon ligand-induced dimerization of the extracellular domain. Although the existence of unliganded FGFR1 dimers on the surface of living cells has been proposed, this notion remains rather controversial. Here, we employed time-resolved Förster resonance energy transfer combined with SNAP- and ACP-tag labeling in COS7 cells to monitor dimerization of full-length FGFR1 at the cell-surface with or without the coreceptor βKlotho. Using this approach we observed homodimerization of unliganded FGFR1 that is independent of its surface density. The homo-interaction signal observed for FGFR1 was indeed as robust as that obtained for epidermal growth factor receptor (EGFR) and was further increased by the addition of activating ligands or pathogenic mutations. Mutational analysis indicated that the kinase and the transmembrane domains, rather than the extracellular domain, mediate the ligand-independent FGFR1 dimerization. In addition, we observed a formation of a higher order ligand-independent complex by the c-spliced isoform of FGFR1 and βKlotho. Collectively, our approach provides novel insights into the assembly and dynamics of the full-length FGFRs on the cell surface.
From the Biochemical and Cellular Pharmacology, Protein Chemistry, and compsagrar.laetitia@gene.com.
Laëtitia
L
Comps-Agrar
Molecular Biology, Genentech, Inc., South San Francisco, California 94080.
Diana Ronai
DR
Dunshee
Protein Chemistry, and.
Dan L
DL
Eaton
Molecular Biology, Genentech, Inc., South San Francisco, California 94080 junichis@gene.com.
Junichiro
J
Sonoda
D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D004797:Enzyme-Linked Immunosorbent Assay; D066246:ErbB Receptors / Q000378:metabolism; D005346:Fibroblast Growth Factors / Q000737:chemistry; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D000090265:Klotho Proteins; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D055503:Protein Multimerization; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000737:chemistry*; D015398:Signal Transduction; D013329:Structure-Activity Relationship
C538995:KLB protein, human; D008024:Ligands; D008565:Membrane Proteins; C414620:fibroblast growth factor 21; D005346:Fibroblast Growth Factors; D066246:ErbB Receptors; C496348:FGFR1 protein, human; D051496:Receptor, Fibroblast Growth Factor, Type 1; D000090265:Klotho Proteins
dimerization; fgf21; fibroblast growth factor (FGF); fibroblast growth factor receptor (FGFR); fluorescence resonance energy transfer (FRET); receptor structure-function; receptor-tyrosine kinase; tr-fret