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

Highlights

• FLT1 cleavage releases an N-terminal ectodomain which antagonizes the effects of VEGF.

• The FLT1 cleavage site is adjacent to the TMD between residues 759 and 763.

• This cleavage site is not dependent on FLT1-specific intracellular residues.

• A second downstream cleavage is not regulated by the upstream ectodomain cleavage.

• Ectodomain cleavage regulates cellular signaling via the p42/44 MAP kinase pathway.

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 (767 V) 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.

Introduction

FLT1, also known as VEGFR1, is one of the two principal cell surface receptors for VEGF and is critically important for angiogenesis not only in development but also during pregnancy and following injury [1], [2]. FLT1 and KDR also known as VEGFR2 are type 1 transmembrane proteins with an extracellular N-terminal region that includes the ligand binding domain, a single transmembrane domain and an intracellular C-terminal region that contains a split tyrosine kinase domain [3], [4]. VEGF and a related growth factor PlGF bind VEGF receptors as a homodimer or heterodimer leading to receptor tyrosine phosphorylation and downstream signaling including the activation of protein kinase C (PKC) and MAP kinases. The activity of the VEGF receptors can be also be regulated by the presence of naturally occurring receptor antagonists. In this regard, several truncated FLT1 variants bind VEGF and PlGF with high affinity reducing free ligand and thus inhibiting receptor function [5]. Two of the soluble FLT1 (sFLT1) variants are transcriptionally derived and prematurely terminate by alternate splicing and utilization of upstream polyadenylation sites to yield secreted proteins that lack the transmembrane and C-terminal domains [6], [7], [8], [9]. FLT1 is also proteolytically cleaved close to the transmembrane domain by ADAM metalloproteases to release the N-terminal fragment into the extracellular milieu [10]. Cleaved FLT1 (cFLT1) like sFLT1 contain the VEGF binding domain and serves as a decoy receptor to reduce VEGF and PlGF access to its cognate cell surface receptors and thus function as VEGF and/or PlGF antagonists.

Proteolytic cleaving of surface proteins is now widely recognized as a mechanism for the release of protein fragments that serve a wide variety of purposes [11], [12]. In some instances, as with FLT1, the release of a soluble receptor antagonist is one mechanism to regulate VEGF function in an autocrine, paracrine or endocrine fashion. In other situations, proteolytic cleaving is used to release proligands such as proHB-EGF and proTGF-α as soluble agonists, or to increase circulating cytokines such as TNF-α or cell adhesion molecules such as selectins and cadherins [13]. One of the common class of ‘sheddases’ are metalloproteases of the ADAM superfamily and individual ADAMs can cleave multiple substrates and the same substrate can be cleaved sometimes by more than one ADAM protease [12]. The extracellular cleavage of membrane proteins do not appear to be determined by a unique signature or common motif within the target protein although the cleavage site is usually close to the TMD and it is unclear if secondary structures in this area or the proximity to the TMD are key determinants of cleavage.

Many extracellular cleavage events are accompanied by a downstream cleavage event that occurs within or just beyond the TMD which releases a fragment internally. This process, called regulated intramembrane proteolysis (RIPS) seems to follow the upstream cleavage event [14], [15], [16]. The internally released fragments may traffic to the nucleus or other intracellular organelles and be involved in transcription or in cellular signaling or be a mechanism to stimulate target protein release, terminate protein function or to effect its degradation. The enzymes that catalyze RIPS are called intramembrane-cleaving proteases (iCLIPS) and generally belong to one of three enzyme families. These are the aspartyl proteases like γ-secretase, the zinc metalloproteinase site-2 proteinase and serine proteases of the rhomboid family [16], [17], [18].

In this manuscript, we further explore the cleavage of FLT1. We identify the site of ectodomain cleavage and demonstrate a second cleavage event that releases a cytosolic fragment. Remarkably, the downstream cleavage event can occur without the preceding upstream cleavage challenging the dogma that ectodomain cleavage is a prerequisite for the intracellular cleavage. This downstream cleavage does not appear to be γ-secretase dependent. We also show that cleavage resistant FLT1 mutants demonstrates lower p44/42 MAP kinase activation compared to wild type FLT1 suggesting that cleavage regulates receptor activation and signaling.