{"id":107407,"date":"2022-08-24T14:50:07","date_gmt":"2022-08-24T18:50:07","guid":{"rendered":"https:\/\/med.virginia.edu\/cell-biology\/?p=107407"},"modified":"2025-05-15T11:34:07","modified_gmt":"2025-05-15T15:34:07","slug":"quantitation-of-rhoa-activation-differential-binding-to-downstream-effectors","status":"publish","type":"post","link":"https:\/\/med.virginia.edu\/cell-biology\/2022\/08\/24\/quantitation-of-rhoa-activation-differential-binding-to-downstream-effectors\/","title":{"rendered":"Quantitation of RhoA activation: differential binding to downstream effectors"},"content":{"rendered":"<p><strong>UVA Author:<\/strong> Yuwen Zhang<br \/>\n<strong>Citation:<\/strong> Zhang YW, Torsilieri HM, Casanova JE. Quantitation of RhoA activation: differential binding to downstream effectors. Small GTPases. 2022 Jan;13(1):296-306. doi: 10.1080\/21541248.2022.2111945. PMID: 35950594; PMCID: PMC9377269.<\/p>\n<p><strong>DOI:<\/strong> <a>https:\/\/doi.org\/10.1080\/21541248.2022.2111945<\/a><br \/>\n<strong>Pub-Med Number<\/strong>: 35950594<\/p>\n<hr \/>\n<div id=\"enc-abstract\" class=\"abstract-content selected\">\n<p>The small GTPase RhoA controls many important cellular processes through its ability to activate multiple downstream effector pathways. Most RhoA effectors contain a Rho-binding domain (RBD), and interaction between active RhoA and the RBD typically induces a conformational change in effectors that stimulates their recruitment or activity. Isolated GTPase binding domains fused to GST have been widely used in so-called pulldown assays to measure the activation state of other GTPases in cell lysates. Similarly, GST fusions containing the RBD of the RhoA effector Rhotekin have been widely adopted as a standardized tool for the measurement of RhoA activation. RBDs have also been used to generate fluorescent reporter constructs to localize sites of GTPase activation in intact cells. In this report, we demonstrate that not all forms of active RhoA are capable of interacting with the Rhotekin RBD. A constitutively active RhoA-G14V mutant, which interacted with the RBDs of ROCK2 and mDIA1, was unable to bind the Rhotekin RBD as evidenced by both conventional GST pulldown assay and our newly established BRET assay. Furthermore, active RhoA induced by different stimuli in cells also displayed binding preference for its diverse effectors. Our data demonstrate that RhoA may undergo effector-specific activation for differential regulation of its downstream pathways, and that RhoA activation should not be defined solely by its interaction with Rhotekin.<\/p>\n<\/div>\n","protected":false},"excerpt":{"rendered":"<p>UVA Author: Yuwen Zhang Citation: Zhang YW, Torsilieri HM, Casanova JE. Quantitation of RhoA activation: differential binding to downstream effectors. Small GTPases. 2022 Jan;13(1):296-306. doi: 10.1080\/21541248.2022.2111945. PMID: 35950594; PMCID: PMC9377269. DOI: https:\/\/doi.org\/10.1080\/21541248.2022.2111945 Pub-Med Number: 35950594 The small GTPase RhoA controls many important cellular processes through its ability to activate multiple downstream effector pathways. Most RhoA [&hellip;]<\/p>\n","protected":false},"author":1740,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"inline_featured_image":false,"footnotes":"","_links_to":"","_links_to_target":""},"categories":[13],"tags":[],"class_list":["post-107407","post","type-post","status-publish","format-standard","hentry","category-featured-publications"],"acf":false,"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v26.1.1 - https:\/\/yoast.com\/wordpress\/plugins\/seo\/ -->\n<title>Quantitation of RhoA activation: differential binding to downstream effectors - Department of Cell Biology<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/med.virginia.edu\/cell-biology\/2022\/08\/24\/quantitation-of-rhoa-activation-differential-binding-to-downstream-effectors\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Quantitation of RhoA activation: differential binding to downstream effectors - Department of Cell Biology\" \/>\n<meta property=\"og:description\" content=\"UVA Author: Yuwen Zhang Citation: Zhang YW, Torsilieri HM, Casanova JE. Quantitation of RhoA activation: differential binding to downstream effectors. Small GTPases. 2022 Jan;13(1):296-306. doi: 10.1080\/21541248.2022.2111945. PMID: 35950594; PMCID: PMC9377269. DOI: https:\/\/doi.org\/10.1080\/21541248.2022.2111945 Pub-Med Number: 35950594 The small GTPase RhoA controls many important cellular processes through its ability to activate multiple downstream effector pathways. Most RhoA [&hellip;]\" \/>\n<meta property=\"og:url\" content=\"https:\/\/med.virginia.edu\/cell-biology\/2022\/08\/24\/quantitation-of-rhoa-activation-differential-binding-to-downstream-effectors\/\" \/>\n<meta property=\"og:site_name\" content=\"Department of Cell Biology\" \/>\n<meta property=\"article:published_time\" content=\"2022-08-24T18:50:07+00:00\" \/>\n<meta property=\"article:modified_time\" content=\"2025-05-15T15:34:07+00:00\" \/>\n<meta name=\"author\" content=\"zkr7e@virginia.edu\" \/>\n<meta name=\"twitter:card\" content=\"summary_large_image\" \/>\n<meta name=\"twitter:label1\" content=\"Written by\" \/>\n\t<meta name=\"twitter:data1\" content=\"zkr7e@virginia.edu\" \/>\n\t<meta name=\"twitter:label2\" content=\"Est. reading time\" \/>\n\t<meta name=\"twitter:data2\" content=\"1 minute\" \/>\n<script type=\"application\/ld+json\" class=\"yoast-schema-graph\">{\"@context\":\"https:\/\/schema.org\",\"@graph\":[{\"@type\":\"WebPage\",\"@id\":\"https:\/\/med.virginia.edu\/cell-biology\/2022\/08\/24\/quantitation-of-rhoa-activation-differential-binding-to-downstream-effectors\/\",\"url\":\"https:\/\/med.virginia.edu\/cell-biology\/2022\/08\/24\/quantitation-of-rhoa-activation-differential-binding-to-downstream-effectors\/\",\"name\":\"Quantitation of RhoA activation: differential binding to downstream effectors - Department of Cell Biology\",\"isPartOf\":{\"@id\":\"https:\/\/med.virginia.edu\/cell-biology\/#website\"},\"datePublished\":\"2022-08-24T18:50:07+00:00\",\"dateModified\":\"2025-05-15T15:34:07+00:00\",\"author\":{\"@id\":\"https:\/\/med.virginia.edu\/cell-biology\/#\/schema\/person\/b4929a1d4683383a392718e12166cd7f\"},\"breadcrumb\":{\"@id\":\"https:\/\/med.virginia.edu\/cell-biology\/2022\/08\/24\/quantitation-of-rhoa-activation-differential-binding-to-downstream-effectors\/#breadcrumb\"},\"inLanguage\":\"en-US\",\"potentialAction\":[{\"@type\":\"ReadAction\",\"target\":[\"https:\/\/med.virginia.edu\/cell-biology\/2022\/08\/24\/quantitation-of-rhoa-activation-differential-binding-to-downstream-effectors\/\"]}]},{\"@type\":\"BreadcrumbList\",\"@id\":\"https:\/\/med.virginia.edu\/cell-biology\/2022\/08\/24\/quantitation-of-rhoa-activation-differential-binding-to-downstream-effectors\/#breadcrumb\",\"itemListElement\":[{\"@type\":\"ListItem\",\"position\":1,\"name\":\"Home\",\"item\":\"https:\/\/med.virginia.edu\/cell-biology\/\"},{\"@type\":\"ListItem\",\"position\":2,\"name\":\"Quantitation of RhoA activation: differential binding to downstream effectors\"}]},{\"@type\":\"WebSite\",\"@id\":\"https:\/\/med.virginia.edu\/cell-biology\/#website\",\"url\":\"https:\/\/med.virginia.edu\/cell-biology\/\",\"name\":\"Department of Cell Biology\",\"description\":\"University of Virginia School of Medicine\",\"potentialAction\":[{\"@type\":\"SearchAction\",\"target\":{\"@type\":\"EntryPoint\",\"urlTemplate\":\"https:\/\/med.virginia.edu\/cell-biology\/?s={search_term_string}\"},\"query-input\":{\"@type\":\"PropertyValueSpecification\",\"valueRequired\":true,\"valueName\":\"search_term_string\"}}],\"inLanguage\":\"en-US\"},{\"@type\":\"Person\",\"@id\":\"https:\/\/med.virginia.edu\/cell-biology\/#\/schema\/person\/b4929a1d4683383a392718e12166cd7f\",\"name\":\"zkr7e@virginia.edu\",\"image\":{\"@type\":\"ImageObject\",\"inLanguage\":\"en-US\",\"@id\":\"https:\/\/med.virginia.edu\/cell-biology\/#\/schema\/person\/image\/\",\"url\":\"https:\/\/secure.gravatar.com\/avatar\/eb05928a9c10d0bc86fc859e223e3e9cd7a535837367cf67c524db2a42f795d0?s=96&d=mm&r=g\",\"contentUrl\":\"https:\/\/secure.gravatar.com\/avatar\/eb05928a9c10d0bc86fc859e223e3e9cd7a535837367cf67c524db2a42f795d0?s=96&d=mm&r=g\",\"caption\":\"zkr7e@virginia.edu\"},\"url\":\"https:\/\/med.virginia.edu\/cell-biology\/author\/zkr7evirginia-edu\/\"}]}<\/script>\n<!-- \/ Yoast SEO plugin. -->","yoast_head_json":{"title":"Quantitation of RhoA activation: differential binding to downstream effectors - Department of Cell Biology","robots":{"index":"index","follow":"follow","max-snippet":"max-snippet:-1","max-image-preview":"max-image-preview:large","max-video-preview":"max-video-preview:-1"},"canonical":"https:\/\/med.virginia.edu\/cell-biology\/2022\/08\/24\/quantitation-of-rhoa-activation-differential-binding-to-downstream-effectors\/","og_locale":"en_US","og_type":"article","og_title":"Quantitation of RhoA activation: differential binding to downstream effectors - Department of Cell Biology","og_description":"UVA Author: Yuwen Zhang Citation: Zhang YW, Torsilieri HM, Casanova JE. 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