Research ArticleEctodomain cleavage of FLT1 regulates receptor activation and function and is not required for its downstream intracellular cleavage
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.
Section snippets
Materials and methods
Heparin, phorbol 12-myristate 13-acetate (PMA), L-685,458, DAPT and Suberic acid bis (N-hydroxy-succinimide ester, DSS) were purchased from Sigma-Aldrich (St. Louis, MO) and Compound E was from EMD Millipore (Billerica, MA). Human VEGF ELISA Kit was obtained from R&D Systems (Minneapolis, MN). Antibodies: HA (Y-11), alkaline phosphatase (sc-28904), α-Tubulin (sc-8035), HSP90 (sc-69703), EGFR (sc-03), Presenilin 1 (sc-7860), HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-mouse
Results
In previous work we have shown that PKC activation stimulates the cleavage of an ectodomain of FLT1 that is mediated via ADAM10 and ADAM17 [10]. The cleaved ectodomain shares substantial identity with transcribed forms of FLT1 that lack both the transmembrane domain and the intracellular portion, and are secreted and are collectively known as soluble FLT1 (sFLT1). Cleaved FLT1, like sFLT1, is heavily glycosylated, can bind VEGF and PlGF and can function as a VEGF antagonist in the extracellular
Discussion
FLT1, like many other type 1 membrane proteins appears to be subject to ectodomain cleavage and a downstream intramembrane or cytosolic cleavage event. We have previously shown that the ectodomain cleavage does not require the presence of the intracellular domain suggesting that receptor activation and downstream signaling is not a prerequisite for cleavage. Ectodomain cleavage of membrane proteins can serve many functions including the release of a bioactive peptide or trigger a downstream
Acknowledgements
We thank the University of Iowa DNA and Vector Core facility for services provided. We thank Dr. Gopal Thinakaran for the generous gift of plasmids and for helpful comments. This work was supported by the National Institutes of Health, RO1 DK090053.
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2018, American Journal of Physiology - Cell Physiology