N-terminal cleavage and release of the ectodomain of Flt1 is mediated via ADAM10 and ADAM 17 and regulated by VEGFR2 and the Flt1 intracellular domain.
Flt is one of the cell surface VEGF receptors which can be cleaved to release an N-terminal extracellular fragment which, like alternately transcribed soluble Flt1 (sFlt1), can antagonize the effects of VEGF. In HUVEC and in HEK293 cells where Flt1 was expressed, metalloprotease inhibitors reduced Flt1 N-terminal cleavage. Overexpression of ADAM10 and ADAM17 increased cleavage while knockdown of ADAM10 and ADAM17 reduced N-terminal cleavage suggesting that these metalloproteases were responsible for Flt1 cleavage. Protein kinase C (PKC) activation increased the abundance and the cleavage of Flt1 but this did not require any residues within the intracellular portion of Flt1. ALLN, a proteasomal inhibitor, increased the abundance of Flt1 which was additive to the effect of PKC. Removal of the entire cytosolic region of Flt1 appeared to stimulate cleavage of Flt1 and Flt1 was no longer sensitive to ALLN suggesting that the cytosolic region contained a degradation domain. Knock down of c-CBL, a ring finger ubiquitin ligase, in HEK293 cells increased the expression of Flt1 although it did not appear to require a previously published tyrosine residue (1333Y) in the C-terminus of Flt1. Increasing VEGFR2 expression increased VEGF-stimulated sFlt1 expression and progressively reduced the cleavage of Flt1 with Flt1 staying bound to VEGFR2 as a heterodimer. Our results imply that secreted sFlt1 and cleaved Flt1 will tend to have local effects as a VEGF antagonist when released from cells expressing VEGFR2 and more distant effects when released from cells lacking VEGFR2.
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA, United States of America.
Nandita S
NS
Raikwar
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA, United States of America.
Kang Z
KZ
Liu
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA, United States of America; Department of Molecular and Cellular Biology, University of Iowa College of Medicine, Iowa City, IA, United States of America; Department of Pediatrics, University of Iowa College of Medicine, Iowa City, IA, United States of America; Department of Obstetrics, University of Iowa College of Medicine, Iowa City, IA, United States of America; Veterans Affairs Medical Center, Iowa City, IA, United States of America.
Christie P
CP
Thomas
D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism*; D000072197:ADAM10 Protein; D000072198:ADAM17 Protein; D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000378:metabolism*; D004151:Dipeptides / Q000494:pharmacology; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D061965:Matrix Metalloproteinase Inhibitors / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D017434:Protein Structure, Tertiary; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism*
D004151:Dipeptides; D006877:Hydroxamic Acids; D007976:Leupeptins; D061965:Matrix Metalloproteinase Inhibitors; D008565:Membrane Proteins; C092152:N-((2-(hydroxyaminocarbonyl)methyl)-4-methylpentanoyl)-3-(2'-naphthyl)alanylalanine, 2-aminoethylamide; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C063261:acetylleucyl-leucyl-norleucinal; C501162:FLT1 protein, human; C000588866:KDR protein, human; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2; D053829:Amyloid Precursor Protein Secretases; D051722:ADAM Proteins; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human