ABSTRACT
The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein alpha subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling. RGS12 and RGS14 contain not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single G alpha(i/o)-Loco (GoLoco) motif predicted to represent a second G alpha interaction site. Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14. Both regions interact exclusively with G alpha(i1), G alpha(i2), and G alpha(i3) in their GDP-bound forms. In GTP gamma S binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by G alpha(i1). Both regions also stabilize G alpha(i1) in its GDP-bound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF(4)(-). Our results indicate that both RGS12 and RGS14 harbor two distinctly different G alpha interaction sites: a previously recognized N-terminal RGS box possessing G alpha(i/o) GAP activity and a C-terminal GoLoco region exhibiting G alpha(i) GDI activity. The presence of two, independent G alpha interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple G alpha GAP activity.
Subject(s)
Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Heterotrimeric GTP-Binding Proteins/metabolism , RGS Proteins/metabolism , Aluminum Compounds/pharmacology , Amino Acid Sequence , Amino Acid Substitution , Animals , Binding Sites , Biosensing Techniques , Cloning, Molecular , Escherichia coli , Fluorides/pharmacology , Guanosine Diphosphate/metabolism , Heterotrimeric GTP-Binding Proteins/chemistry , Kinetics , Models, Biological , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligopeptides , Open Reading Frames , RGS Proteins/chemistry , RGS Proteins/genetics , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Signal Transduction , Surface Plasmon ResonanceSubject(s)
RGS Proteins/chemistry , RGS Proteins/classification , Amino Acid Motifs , Amino Acid Sequence , Animals , GTP-Binding Proteins/metabolism , Humans , Mammals , Molecular Sequence Data , Multigene Family/genetics , Phylogeny , RGS Proteins/genetics , RGS Proteins/metabolism , Signal TransductionABSTRACT
Members of the newly described RGS family of proteins have a common RGS domain that contains GTPase-activating activity for many Galpha subunits of heterotrimeric G proteins. Their ability to dampen signalling via Galphai-, Galphaq- and Galpha12/13-coupled pathways makes them crucial players in mediating the multitude of cellular processes controlled by heterotrimeric G proteins. Some RGS proteins also contain additional motifs that link them to other signalling networks, where they constitute effector-type molecules. This review summarizes recent findings on RGS proteins, especially those that implicate RGS proteins in more than just enhancing the GTPase activity of their Galpha subunit targets.
