Interrogating GTPase Effector Proteins: Screening GAPs and GEFs with the Transcreener GDP Assay

GTPases control diverse cellular processes by cycling between inactive GDP- and active GTP-bound conformations.  Though they are clearly involved in many diseases (e.g., KRAS in cancer), development of GTP-competitive inhibitors is extremely challenging for a number of reasons.  Targeting the effector proteins that control the activation state of GTPases is a logical alternative approach.  Toward this end, we have developed HTS-compatible assay methods for detection of GTPase-accelerating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) based on their ability to stimulate steady-state GTPase activity under appropriate conditions.

In this webinar, we will describe:

  • How the Transcreener GDP Assay enables robust screening and profiling of GTPases using direct immunodetection of GDP with fluorescence polarization (FP), fluorescence intensity (FI) or TR-FRET readouts.
  • How to measure GTPase-GEF catalytic activity based on stimulation of steady state GDP formation, using the Transcreener GDP Assay. Examples of two DOCK family Rho GEFs, Dbl and P-Rex1 with their respective Rho GTPase substrates, RhoA, CDC42 and P-Rex1 will be used as case studies.
  • Development of a robust HTS method for identifying inhibitors of members of the “regulator of G-protein signaling” (RGS)-protein superfamily that act as GAPs for heterotrimeric G-protein alpha subunits, using Transcreener® GDP assay.
Interrogating GTPase Effector Proteins Screening GAPs and GEFs with the Transcreener® GDP Assay

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