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RGS12 and RGS14 GoLoco motifs are G alpha(i) interaction sites with guanine nucleotide dissociation inhibitor
R J Kimple1, L De Vries, H Tronchère
1Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7365, USA.
The Journal of Biological Chemistry
|June 2, 2001
Summary
Regulators of G-protein signaling (RGS) proteins RGS12 and RGS14 possess two distinct G alpha interaction sites. Their GoLoco motif acts as a guanine nucleotide dissociation inhibitor (GDI), complementing the RGS box's GTPase-accelerating activity.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Regulators of G-protein signaling (RGS) proteins modulate G-protein-coupled receptor (GPCR) signaling by accelerating GTPase activity of G-protein alpha subunits.
- RGS12 and RGS14 are unique RGS proteins containing both an RGS box and a GoLoco motif, suggesting dual interaction sites with G proteins.
Purpose of the Study:
- To functionally characterize the GoLoco motif regions of RGS12 and RGS14.
- To elucidate the specific interactions and activities mediated by the GoLoco motifs of RGS12 and RGS14 with G alpha subunits.
Main Methods:
- Guanosine diphosphate (GDP)-bound G alpha(i1) interaction assays.
- Guanosine triphosphate gamma S (GTPγS) binding assays to assess guanine nucleotide dissociation inhibitor (GDI) activity.
- Aluminium fluoride (AlF4-) stimulated tryptophan fluorescence assays to evaluate G alpha(i1) stabilization.
Main Results:
- The GoLoco regions of RGS12 and RGS14 exclusively interact with GDP-bound G alpha(i1), G alpha(i2), and G alpha(i3).
- Both GoLoco regions demonstrated GDI activity, significantly inhibiting the rate of GDP to GTP exchange on G alpha(i1).
- These regions also stabilized G alpha(i1) in its GDP-bound state, preventing conformational changes indicated by fluorescence.
Conclusions:
- RGS12 and RGS14 possess two independent G alpha interaction sites: the N-terminal RGS box with GTPase-accelerating protein (GAP) activity and the C-terminal GoLoco motif with GDI activity.
- The dual interaction sites suggest a complex regulatory role for RGS12 and RGS14 in coordinating G-protein signaling pathways beyond simple GAP function.