Antibody-induced dimerization of FGFR1 promotes receptor endocytosis independently of its kinase activity

Abstract

Fibroblast growth factors (FGFs) and their plasma membrane-localized receptors (FGFRs) play a key role in the regulation of developmental processes and metabolism. Aberrant FGFR signaling is associated with the progression of serious metabolic diseases and human cancer. Binding of FGFs to FGFRs induces receptor dimerization and transphosphorylation of FGFR kinase domains that triggers activation of intracellular signaling pathways. Following activation, FGFRs undergo internalization and subsequent lysosomal degradation, which terminates transmission of signals. Although factors that regulate FGFR endocytosis are continuously discovered, little is known about the molecular mechanism that initiates the internalization of FGFRs. Here, we analyzed the internalization of antibody fragments in various formats that target FGFR1. We show that FGFR1-specific antibody fragments in the monovalent scFv format bind to FGFR1, but are not internalized into cells that overproduce FGFR1. In contrast, the same scFv proteins in the bivalent scFv-Fc format are efficiently internalized via FGFR1-mediated, clathrin and dynamin dependent endocytosis. Interestingly, the receptor tyrosine kinase activity is dispensable for endocytosis of scFv-Fc-FGFR1 complexes, suggesting that only dimerization of receptor is required to trigger endocytosis of FGFR1 complexes.

FGFR1 clustering with engineered tetravalent antibody improves the efficiency and modifies the mechanism of receptor internalization

“…Fully glycosylated extracellular domains of FGFRs fused to the Fc fragment of human IgG1: FGFR1 IIIc (FGFR1‐Fc), FGFR2 IIIc (FGFR2‐Fc), FGFR3 IIIc (FGFR3‐Fc), and FGFR4 (FGFR4‐Fc), and the extracellular part of FGFR1 lacking the N‐terminal D1 domain (FGFR1 D2‐D3) were produced as described previously by our group [48]. FGFR1 GST‐D1 was expressed in E. coli BL21 CodonPlus (DE3)‐RIL (Agilent Technologies, Santa Clara, CA, USA) and purified with use of Glutathione Sepharose column as described previously [5,6,49].…”

“…The extracellular domain of FGFR1 contains three immunoglobulin‐like domains D1, D2, and D3. The D1 domain fulfills a regulatory function preventing FGFR1 from autoactivation in the absence of FGFs [3–6]. The D2 and D3 domains form FGF binding sites [1].…”

“…The uptake of FGF/FGFR1 complexes mainly occurs via clathrin‐mediated endocytosis (CME) leading to lysosomal degradation of FGFR1 [29,30,36,37,39]. We have recently uncoupled FGFR1 dimerization from the receptor activation and have demonstrated that dimerization of FGFR1, but not receptor autophosphorylation, triggers CME of FGFR1 [5,6]. A number of proteins involved in CME of FGFR1 in response to FGF binding were identified,however, the exact role of a number of these factors in the receptor internalization is still largely mysterious [31,40–44].…”

Differential regulation of fibroblast growth factor receptor 1 trafficking and function by extracellular galectins

“…1a). Growth factor binding leads to FGFR1 trans-activation and effectively stimulates internalization of FGF-FGFR1 complexes [17,46,47]. To study whether FGF1 binding to SBP-FGFR1 accelerates uptake of the receptor, U2OS-SBP-R1 cells were pre-labeled with streptavidin-AF555 and then incubated in the presence or absence of FGF1.…”

“…1d, lanes 5 and 6). We and others have recently demonstrated that in the absence of FGF1 FGFR1 is present in high molecular weight complexes that likely represent unliganded receptor dimers [47][48][49][50][51]. To study whether isolated SBP-FGFR1 retained its ability to form high molecular weight complexes we have performed streptavidin-agarose pull down and eluted bound proteins with biotin.…”