Ectodomain cleavage of FLT1 regulates receptor activation and function and is not required for its downstream intracellular cleavage

Abstract

Flt1 is a cell surface VEGF receptor which is cleaved to release an N-terminal ectodomain which binds VEGF and PlGF and can antagonize the effects of VEGF in the extracellular milieu. To further evaluate Flt1 processing we expressed tagged Flt1 constructs in HEK293 and COS7 cells where we demonstrate, by deletion mapping, that the cleavage site is immediately adjacent to the transmembrane domain (TMD) between residues 759 and 763. Cleavage reciprocally regulates free VEGF in conditioned media and we show that the cleavage site is also transferable to another transmembrane receptor. A second cleavage event downstream of the ectodomain cleavage releases a cytosolic C-terminal Flt1 fragment and this intracellular cleavage of Flt1 is not catalyzed or regulated by the upstream ectodomain cleavage since abolition of the ectodomain cleavage has no impact on the downstream cleavage event. The downstream cleavage event is not susceptible to γ-secretase inhibitors and overexpression of presenilin 1, the catalytic subunit of γ-secretase did not change the downstream intracellular cleavage event. Furthermore, this cleavage did not occur via a previously published valine residue (767V) in the TMD of Flt1, indicating the existence of another cleavage pathway. We tested the impact of the ectodomain cleavage on p44/42 MAP kinase activation and demonstrate that compared to wild type Flt1, cleavage resistant Flt1 constructs failed to stimulate p44/42 MAP kinase activation. Our results indicate that Flt1 ectodomain cleavage not only regulates the availability of free VEGF in the extracellular milieu but also regulates cellular signaling via the ERK kinase pathway.

VEGF-A selectively inhibits FLT1 ectodomain shedding independent of receptor activation and receptor endocytosis

“…We have previously reported that the single-pass membrane receptor FLT1 is cleaved twice: one cleavage occurs in the extracellular region to shed an NH 2 -terminal fragment (NTF; cFLT1) into the extracellular milieu, leaving a membranetethered COOH-terminal fragment (CTF1), and another cleavage occurs independently within or just after the transmembrane domain to release a cytosolic COOH-terminal fragment, CTF2 (21). Extracellular cleavage is mediated by ADAM10 and ADAM17 and stimulated by protein kinase C (PKC), and the cleaved fragment of FLT1 (cFLT1) binds VEGF-A and PlGF with high affinity and thus functions as a soluble PlGF/ VEGF antagonist regulating angiogenesis in vitro (22,23).…”

“…Expression vectors. Expression vector carrying cDNA for HA-FLT1-Myc-Flag, FLT1⌬CTD, FLT1⌬729 -758, and FLT1Y1333F have been described before (21)(22)(23). COOH-terminal turbo-GFPtagged open reading frame clone of full-length human VEGFR2 (KDR) was purchased from OriGene.…”

“…Secreted proteins in CM were concentrated with Amicon Ultra centrifugal filters from EMD Millipore, and cell lysates were prepared in radioimmunoprecipitation assay buffer (Pierce, Rockford, IL) supplemented with protease inhibitor cocktail from Roche Applied Science (Indianapolis, IN). Total proteins prepared in radioimmunoprecipitation assay buffer were quantitated with BCA Protein Assay Kit from Pierce, and cytosolic and membrane protein fractions were prepared using ProteoExtract Subcellular Proteome Extraction Kit (EMD Millipore) as previously described (21). HSP90 and EGFR, respec-tively, were used as loading controls in immunoblots of cytosolic and membrane protein fractions, based on their abundance and restriction to those subcellular fractions.…”

“…In some cases, protein samples were run on SDS-PAGE under nonreducing conditions by mixing with 2X Laemmli buffer without DTT. After transfer, membranes were incubated with various primary and secondary antibodies, and immunoblotting was performed as previously described (21). Chemiluminescent reactions were performed using SuperSignal West Pico, Dura, or Femto Chemiluminescent Substrate (Thermo Fisher Scientific, Rockford, IL) and signals imaged and quantitated using the EC 3 imaging system from UVP, LLC (Upland, CA) and VisionWorks LS image acquisition and analysis software.…”

“…This ectodomain, like sFLT1, can bind VEGF-A, regulate free VEGF-A available for cell surface receptor signaling, and function as a natural VEGF-A antagonist (23). We have identified the FLT1 extracellular cleavage site, shown that a second downstream cleavage releases an intracellular cytosolic fragment, and demonstrated that this intracellular cleavage is not dependent on the extracellular cleavage event (21). Furthermore, we demonstrated that extracellular cleavage-resistant FLT1 mutants show lower p44/42 MAP kinase activation compared with wild-type FLT1, suggesting that cleavage regulates receptor activation and signaling (21).…”