Determination of the contact energies between a regulator of G protein signaling and G protein subunits and phospholipase C beta 1.
Cell signaling proteins may form functional complexes that are capable of rapid signal turnover. These contacts may be stabilized by either scaffolding proteins or multiple interactions between members of the complex. In this study, we have determined the affinities between a regulator of G protein signaling protein, RGS4, and three members of the G protein-phospholipase Cbeta (PLC-beta) signaling cascade which may allow for rapid deactivation of intracellular Ca(2+) release and activation of protein kinase C. Specifically, using fluorescence methods, we have determined the interaction energies between the RGS4, PLC-beta, G-betagamma, and both deactivated (GDP-bound) and activated (GTPgammaS-bound) Galpha(q). We find that RGS4 not only binds to activated Galpha(q), as predicted, but also to Gbetagamma and PLCbeta(1). These interactions occur through protein-protein contacts since the intrinsic membrane affinity of RGS4 was found to be very weak in the absence of the protein partner PLCbeta(1) or a lipid regulator, phosphatidylinositol-3,4,5 trisphosphate. Ternary complexes between Galpha(q), Gbetagamma and phospholipase Cbeta(1) will form, but only at relatively high protein concentrations. We propose that these interactions allow RGS4 to remain anchored to the signaling complex even in the quiescent state and allow rapid transfer to activated Galpha(q) to shut down the signal. Comparison of the relative affinities between these interacting proteins will ultimately allow us to determine whether certain complexes can form and where signals will be directed.
Dowal
L
L
Department of Physiology & Biophysics, State University of New York at Stony Brook, Stony Brook, New York 11794-8661, USA.
Elliott
J
J
Popov
S
S
Wilkie
T M
TM
Scarlata
S
S
D000818:Animals; D004735:Energy Transfer; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D010716:Phosphatidylinositols / Q000378:metabolism; D054799:Phospholipase C beta; D011485:Protein Binding / Q000235:genetics; D020710:RGS Proteins / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D018411:Spodoptera / Q000235:genetics; D013816:Thermodynamics; D010738:Type C Phospholipases / Q000737:chemistry* / Q000378:metabolism
D007527:Isoenzymes; D008081:Liposomes; D046911:Macromolecular Substances; D010716:Phosphatidylinositols; D020710:RGS Proteins; C118303:phosphoinositide-3,4,5-triphosphate; C098799:RGS4 protein; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins