{"id":1011,"date":"2026-06-13T15:13:25","date_gmt":"2026-06-13T15:13:25","guid":{"rendered":"http:\/\/changingfaceofamerica.com\/?p=1011"},"modified":"2026-06-13T15:13:25","modified_gmt":"2026-06-13T15:13:25","slug":"reduced-vegfr2-availability-at-the-plasma-membrane-could-thus-diminish-response-to-exogenously-added-vegfa","status":"publish","type":"post","link":"https:\/\/changingfaceofamerica.com\/?p=1011","title":{"rendered":"\ufeffReduced VEGFR2 availability at the plasma membrane could thus diminish response to exogenously added VEGFA"},"content":{"rendered":"<p>\ufeffReduced VEGFR2 availability at the plasma membrane could thus diminish response to exogenously added VEGFA. dimerization and transautophosphorylation of several important tyrosine residues present within its cytoplasmic kinase domain1. Upon activation, VEGFR2 enters the endosomelysosome system through incorporation into clathrincoated vesicles and trafficking to early endosomal vesicular compartments4. Ubiquitination of VEGFR2 acts as an endosomal sorting signal by binding to the ubiquitininteracting motif of ESCRT0 components, Hrs and STAM4, 5, 6. Internalized VEGFR2 can recycle back to the plasma membrane or be committed to get lysosomal degradation6, 7. Ubiquitination is a powerful protein customization that coordinates receptor trafficking, recycling SRPKIN-1 and degradation8. Reversibilty of ubiquitination is credited to the action of deubiquitinating enzymes (DUBs)8. These enzymes thus play a distinct but crucial role in receptor tyrosine kinase trafficking and turnover8. DUBs are a superfamily of 91 enzymes that can be subdivided into five unique subfamilies with differing specificities for the isopeptide relationship that links ubiquitin chains9. Deubiquitination of plasma membrane receptors facilitates recycling and is essential for maintaining the totally free ubiquitin pool upon which receptor trafficking is dependent. Similar to the coordinated but opposition effects of kinase and phosphatase activity, ubiquitination is held in balance by the activity <a href=\"http:\/\/webexhibits.org\/causesofcolor\/2.html#vissamp\"> FRP-2<\/a> of DUBs10. Although it is known that VEGFR2 is usually recycled coming from endosomes back to the plasma membrane, it is unknown which DUBs prevent its lysosomal degradation. Ubiquitinspecific protease Y (UBPY or USP8) is actually a DUB be involved in the trafficking of epidermal growth receptor tyrosine kinase (EGFR)11, 12, 13. USP8 is a cysteine protease and member of the ubiquitinspecific protease (UBP) family of DUB enzymes capable of catalyzing total breakdown of both K48 and K63linked polyubiquitin into its component monomers11, 14, 15. USP8 offers diverse SRPKIN-1 roles in membrane trafficking ranging from endosomal regulation to retrograde transport11, 13. The early endosome ESCRT0 subunit, STAM, is actually a USP8binding partner16, 17. This interaction happens via the SH3 domain of STAM and the prolinerich STAMbinding motif in USP817. USP8 depletion inhibits EGFR degradation and causes build up of ubiquitinated proteins on enlarged endosomes11, 12. Once internalized freight has been committed for degradation, conjugated ubiquitin must be recycled and removed by endosomal DUBs such as USP8, which also connect with the ESCRTIII complex on late endosomes8, 18. A model was proposed in which USP8 acts further downstream of early endosomes to recycle ubiquitin after endosomal sorting and prior to lysosomal sequestration, suggesting a role in facilitating membrane receptor degradation14. USP8 thus functions at two stages of plasma membrane receptor trafficking: in early endosomes via ESCRT0 interaction or in late endosomes via ESCRTIII interaction. In this study, we show that regulation of VEGFR2 trafficking and deubiquitination by USP8 effects on downstream signal transduction and proteolysis. == Results == == USP8 regulates VEGFR2 trafficking == Previous studies have shown that USP8 depletion causes EGFR build up in early endosomes and inhibits downstream degradation due to general defects in endosomal sorting11. USP8 thus seemed a likely candidate to get regulating VEGFR2 trafficking. To test this, we used siRNA duplexes to deplete USP8 in main human endothelial cells prior to VEGFA activation and immunofluorescence analysis (Figure1A). In control cells treated with nontargeting siRNA, internalized VEGFR2 was detected in punctate structures at early (015 min) stages of VEGFA stimulation (Figure1A). After VEGFA stimulation to get 60 min, VEGFR2 staining was substantially reduced SRPKIN-1 consistent with ligandinduced degradation <a href=\"https:\/\/www.adooq.com\/srpkin-1.html\">SRPKIN-1<\/a> (Figure1A). == Figure 1 . == USP8 is essential to get VEGFR2 trafficking. A) Endothelial cells transfected with nontargeting or USP8 siRNA, pretreated with CHX and stimulated with 25 ng\/mL VEGFA were fixed and processed for immunofluorescence microscopy using antibodies to VEGFR2 followed by fluorescent speciesspecific secondary antibodies (green). Nuclei were stained with DNAbinding dye, DAPI (blue). Level bar represents 200 m. B) Quantification of VEGFR2 levels residing in the endosomelysosome system in endothelial cells pretreated with CHX and stimulated with VEGFA prior to immunofluorescence analysis. C) Endothelial cells SRPKIN-1 transfected with USP8 siRNA and stimulated with 25 ng\/mL VEGFA to get 15 min were fixed and processed for immunofluorescence microscopy using antibodies to VEGFR2 (green) and EEA1 (red) followed by fluorescent speciesspecific secondary antibodies. Nuclei were stained with DNAbinding dye, DAPI (blue). Scale club represents 70 m. D) Quantification of codistribution between VEGFR2 and EEA1 in endothelial cells treated with nontargeting or USP8 siRNA and stimulated with VEGFA prior to immunofluorescence analysis. Errors bars indicated SEM (n 3); p < 0. 05 (*), p < 0. 0001 (****). However , in USP8depleted.\n<\/p>\n","protected":false},"excerpt":{"rendered":"<p>\ufeffReduced VEGFR2 availability at the plasma membrane could thus diminish response to exogenously added VEGFA. dimerization and transautophosphorylation of several important tyrosine residues present within its cytoplasmic kinase domain1. Upon activation, VEGFR2 enters the endosomelysosome system through incorporation into clathrincoated vesicles and trafficking to early endosomal vesicular compartments4. Ubiquitination of VEGFR2 acts as an endosomal [&hellip;]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[30],"tags":[],"class_list":["post-1011","post","type-post","status-publish","format-standard","hentry","category-glutamate-nmda-receptors"],"_links":{"self":[{"href":"https:\/\/changingfaceofamerica.com\/index.php?rest_route=\/wp\/v2\/posts\/1011","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/changingfaceofamerica.com\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/changingfaceofamerica.com\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/changingfaceofamerica.com\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/changingfaceofamerica.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=1011"}],"version-history":[{"count":1,"href":"https:\/\/changingfaceofamerica.com\/index.php?rest_route=\/wp\/v2\/posts\/1011\/revisions"}],"predecessor-version":[{"id":1012,"href":"https:\/\/changingfaceofamerica.com\/index.php?rest_route=\/wp\/v2\/posts\/1011\/revisions\/1012"}],"wp:attachment":[{"href":"https:\/\/changingfaceofamerica.com\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=1011"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/changingfaceofamerica.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=1011"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/changingfaceofamerica.com\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=1011"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}