Identification of guanine nucleotide exchange factors that increase Cdc42 activity in primary human endothelial cells

Small GTPases. 2021 May;12(3):226-240. doi: 10.1080/21541248.2019.1658509. Epub 2019 Aug 30.

Abstract

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.

Keywords: Cdc42; FRET biosensor; Rac1; Rho GEF; endothelial cells; fluorescent protein; guanine exchange factor; image analysis.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / metabolism*
  • Fluorescence Resonance Energy Transfer / methods*
  • Guanine Nucleotide Exchange Factors / metabolism*
  • Human Umbilical Vein Endothelial Cells
  • Humans
  • Models, Molecular
  • Protein Binding
  • Sequence Homology, Amino Acid
  • Single-Cell Analysis / methods
  • cdc42 GTP-Binding Protein / metabolism*
  • rac1 GTP-Binding Protein / metabolism*

Substances

  • Guanine Nucleotide Exchange Factors
  • RAC1 protein, human
  • CDC42 protein, human
  • cdc42 GTP-Binding Protein
  • rac1 GTP-Binding Protein