A direct fluorescence-based assay for RGS domain GTPase accelerating activity.

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Physiology

Background:

• Seven transmembrane-spanning receptors (GPCRs) mediate cellular responses to extracellular signals.
• GPCRs activate heterotrimeric guanine nucleotide binding proteins (G proteins) for signal transduction.
• G protein signaling is regulated by the GTPase activity of Galpha subunits, which is accelerated by regulator of G protein signaling (RGS) proteins.

Purpose of the Study:

• To develop a novel spectroscopic probe for measuring G protein GTPase activity.
• To utilize the probe for real-time analysis of Galphao nucleotide hydrolysis.
• To assess the probe's utility in studying RGS protein function and identifying inhibitors.

Main Methods:

• Synthesis and characterization of BODIPYFL-GTP, a fluorescent guanine nucleotide analog.
• Spectroscopic measurement of BODIPYFL-GTP binding and hydrolysis by Galphao.
• Assay development for real-time monitoring of RGS protein-catalyzed GTP hydrolysis.

Main Results:

• BODIPYFL-GTP exhibits a significant fluorescence increase upon binding to Galphao.
• GTP hydrolysis to GDP results in a decrease in fluorescence quantum yield.
• The assay successfully measured intrinsic and RGS12-catalyzed GTP hydrolysis by Galphao, including analysis of mutants.

Conclusions:

• BODIPYFL-GTP serves as an effective real-time probe for Galphao GTPase activity.
• This assay facilitates the analysis of G protein signaling and RGS protein function.
• The developed assay is valuable for screening RGS protein inhibitors.