Kinetic approaches to study the function of RGS9 isoforms.
The experimental strategies developed in kinetic studies of interactions between RGS9 isoforms with G proteins of the Gi subfamily provide a useful framework for conducting similar studies with essentially any regulator of G-protein signaling (RGS) protein-G-protein pair. This article describes two major kinetic approaches used in the studies of RGS9 isoforms: single turnover and multiple turnover GTPase assays. We also describe pull-down assays as a method complementary to the kinetic assays. The discussion of the strengths and limitations of each individual assay emphasizes the importance of combining multiple experimental approaches in order to obtain comprehensive and internally consistent information regarding the mechanisms of RGS protein action.
Howe Laboratory of Ophthalmology, Harvard Medical School and Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114, USA.
Kirill A
KA
Martemyanov
Vadim Y
VY
Arshavsky
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism; D017398:Alternative Splicing; D000818:Animals; D001681:Biological Assay / Q000379:methods; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D020558:GTP Phosphohydrolases / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000737:chemistry / Q000378:metabolism; D015293:Transducin / Q000378:metabolism
D044385:GTP-Binding Protein alpha Subunits; D020033:Protein Isoforms; D020710:RGS Proteins; C116925:regulator of g-protein signaling 9; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D020558:GTP Phosphohydrolases; D015293:Transducin