Fluorescence-based assay probing regulator of G protein signaling partner proteins.
The regulator of G protein signaling (RGS) proteins are one of the essential modulators for the G protein system. Besides regulating G protein signaling by accelerating the GTPase activity of Gα subunits, RGS proteins are implicated in exerting other functions; they are also known to be involved in several diseases. Moreover, the existence of a single RGS protein in plants and its seven-transmembrane domain found in 2003 triggered efforts to unveil detailed structural and functional information of RGS proteins. We present a method for real-time examination of the protein-protein interactions between RGS and Gα subunits. AtRGS1 from plants and RGS4 from mammals were site-directedly labeled with the fluorescent probe Lucifer yellow on engineered cysteine residues and used to interact with different Gα subunits. The physical interactions can be revealed by monitoring the real-time fluorescence changes (8.6% fluorescence increase in mammals and 27.6% in plants); their correlations to functional exertion were shown with a GTPase accelerating activity assay and further confirmed by measurement of K(d). We validate the effectiveness of this method and suggest its application to the exploration of more RGS signaling partner proteins in physiological and pathological studies.
Department of Biochemical Science and Technology, College of Life Science, National Taiwan University, Taipei 10617, Taiwan.
Po-Shiun
PS
Huang
Hsin-Sung
HS
Yeh
Hsiu-Ping
HP
Yi
Chain-Jia
CJ
Lin
Chii-Shen
CS
Yang
D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001896:Boron Compounds / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism*; D007546:Isoquinolines / Q000737:chemistry; D007700:Kinetics; D025941:Protein Interaction Mapping; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D029681:Arabidopsis Proteins; D001896:Boron Compounds; D005456:Fluorescent Dyes; D044385:GTP-Binding Protein alpha Subunits; D007546:Isoquinolines; D020710:RGS Proteins; D011994:Recombinant Proteins; C017475:lucifer yellow; D003545:Cysteine