Biochemical and electrophysiological analyses of RGS8 function.
RGS8 was identified as a regulator of G-protein signaling (RGS) protein induced in neuronally differentiated P19 cells. This article presents methods used to estimate the binding activity and selectivity between RGS8 and Galpha subunits. It describes three kinds of in vitro-binding experiments using RGS8 proteins generated by three different techniques: recombinant protein from Escherichia coli, in vitro-translated protein, and protein expressed in cDNA-transfected cultured cells. It also presents methods of the functional analysis of RGS protein using the Xenopus oocyte expression system. Electrophysiological procedures, which were used to examine the effects of RGS8 on Gi- and Gq-mediated responses, are described.
Saitoh
Osamu
O
Department of Molecular Cell Signaling, Tokyo Metropolitan Institute for Neuroscience, Japan.
Yoshihiro
Kubo
K
D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D004594:Electrophysiology; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism*; D043643:Receptor, Muscarinic M1 / Q000378:metabolism; D018040:Receptors, Neurokinin-1 / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D044385:GTP-Binding Protein alpha Subunits; D020710:RGS Proteins; D043643:Receptor, Muscarinic M1; D018040:Receptors, Neurokinin-1; D011994:Recombinant Proteins