TY - JOUR
T1 - Identification of guanine nucleotide exchange factors that increase Cdc42 activity in primary human endothelial cells
AU - Reinhard, Nathalie R.
AU - Van Der Niet, Sanne
AU - Chertkova, Anna
AU - Postma, Marten
AU - Hordijk, Peter L.
AU - Gadella, Theodorus W.J.
AU - Goedhart, Joachim
AU - Gadella Jr, Theodorus W. J.
N1 - Funding Information: We thank D. Webb (Vanderbilt University), H.C. Welch (Babraham Institute), J. van Buul (Sanquin Research), J.L. Zugaza (University of the Basque Country), M. Hayakawa (Tokyo University of Pharmacy and Life Sciences), W.J. Pannekoek (University Medical centre Utrecht), I.G. Macara (Vanderbilt University), K. Mizuno (Tohoku University), H. Ueda (Gifu university), D. Manor Case Western Reserve University, and L.J. Bruurs (University Medical centre Utrecht) for providing us with the FL GEF constructs. Publisher Copyright: © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
PY - 2021
Y1 - 2021
N2 - The Rho GTPase family is involved in actin dynamics and regulates the barrier function of the endothelium. One of the main barrier-promoting Rho GTPases is Cdc42, also known as cell division control protein 42 homolog. Currently, regulation of Cdc42-based signalling networks in endothelial cells (ECs) lack molecular details. To examine these, we focused on a subset of 15 Rho guanine nucleotide exchange factors (GEFs), which are expressed in the endothelium. By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1. A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the overexpressed full-length GEFs. Our data reveal GEF dependent activation of Cdc42, with the most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and PREX1 and the highest selectivity for FGD1. Additionally, we generated truncated GEF constructs that comprise only the catalytic dbl homology (DH) domain or together with the adjacent pleckstrin homology domain (DHPH). The DH domain by itself did not activate Cdc42, whereas the DHPH domain of ITSN1, ITSN2 and PLEKHG1 showed activity towards Cdc42. Together, our study characterized endothelial GEFs that may directly or indirectly activate Cdc42, which will be of great value for the field of vascular biology.
AB - The Rho GTPase family is involved in actin dynamics and regulates the barrier function of the endothelium. One of the main barrier-promoting Rho GTPases is Cdc42, also known as cell division control protein 42 homolog. Currently, regulation of Cdc42-based signalling networks in endothelial cells (ECs) lack molecular details. To examine these, we focused on a subset of 15 Rho guanine nucleotide exchange factors (GEFs), which are expressed in the endothelium. By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1. A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the overexpressed full-length GEFs. Our data reveal GEF dependent activation of Cdc42, with the most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and PREX1 and the highest selectivity for FGD1. Additionally, we generated truncated GEF constructs that comprise only the catalytic dbl homology (DH) domain or together with the adjacent pleckstrin homology domain (DHPH). The DH domain by itself did not activate Cdc42, whereas the DHPH domain of ITSN1, ITSN2 and PLEKHG1 showed activity towards Cdc42. Together, our study characterized endothelial GEFs that may directly or indirectly activate Cdc42, which will be of great value for the field of vascular biology.
KW - Cdc42
KW - FRET biosensor
KW - Rac1
KW - Rho GEF
KW - endothelial cells
KW - fluorescent protein
KW - guanine exchange factor
KW - image analysis
UR - http://www.scopus.com/inward/record.url?scp=85071362224&partnerID=8YFLogxK
UR - https://pure.uva.nl/ws/files/62305587/ksgt_a_1658509_sm3535.docx
U2 - https://doi.org/10.1080/21541248.2019.1658509
DO - https://doi.org/10.1080/21541248.2019.1658509
M3 - Article
C2 - 31469028
SN - 2154-1248
VL - 12
SP - 226
EP - 240
JO - Small GTPases
JF - Small GTPases
IS - 3
M1 - DOI: 10.1080/21541248.2019.1658509
ER -