Receptor-regulated interaction of activator of G-protein signaling-4 and Galphai.
Activator of G-protein signaling-4 (AGS4), via its three G-protein regulatory motifs, is well positioned to modulate G-protein signal processing by virtue of its ability to bind Galpha(i)-GDP subunits free of Gbetagamma. Apart from initial observations on the biochemical activity of the G-protein regulatory motifs of AGS4, very little is known about the nature of the AGS4-G-protein interaction, how this interaction is regulated, or where the interaction takes place. As an initial approach to these questions, we evaluated the interaction of AGS4 with Galpha(i1) in living cells using bioluminescence resonance energy transfer (BRET). AGS4 and Galpha(i1) reciprocally tagged with either Renilla luciferase (RLuc) or yellow fluorescent protein (YFP) demonstrated saturable, specific BRET signals. BRET signals observed between AGS4-RLuc and Galpha(i1)-YFP were reduced by G-protein-coupled receptor activation, and this agonist-induced reduction in BRET was blocked by pertussis toxin. In addition, specific BRET signals were observed for AGS4-RLuc and alpha(2)-adrenergic receptor-Venus, which were Galpha(i)-dependent and reduced by agonist, indicating that AGS4-Galpha(i) complexes are receptor-proximal. These data suggest that AGS4-Galpha(i) complexes directly couple to a G-protein-coupled receptor and may serve as substrates for agonist-induced G-protein activation.
Oner
Sukru Sadik
SS
From the Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, SC 29425, USA.
Maher
Ellen M
EM
Breton
Billy
B
Bouvier
Michel
M
Blumer
Joe B
JB
D000818:Animals; D002460:Cell Line; D020555:GTP-Binding Protein Regulators / Q000502:physiology*; D017930:Genes, Reporter; D006706:Homeostasis; D006801:Humans; D007668:Kidney; D008156:Luciferases / Q000235:genetics; D008164:Luminescent Proteins / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism*; D018341:Receptors, Adrenergic, alpha-2 / Q000502:physiology; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D048790:Renilla / Q000201:enzymology; D014162:Transfection
D020555:GTP-Binding Protein Regulators; D008164:Luminescent Proteins; D020710:RGS Proteins; D018341:Receptors, Adrenergic, alpha-2; D043562:Receptors, G-Protein-Coupled; C098799:RGS4 protein; D008156:Luciferases
Proc Natl Acad Sci U S A. 2005 Jun 14;102(24):8746-51
15937104
Nat Struct Mol Biol. 2006 Sep;13(9):778-86
16906158
J Biol Chem. 2004 Jun 25;279(26):27567-74
15096500
J Biol Chem. 2001 Jan 12;276(2):1585-93
11042168
J Biol Chem. 2003 Dec 19;278(51):51825-32
14530282
Proc Natl Acad Sci U S A. 2007 Nov 13;104(46):18103-8
17991770
Br J Pharmacol. 2006 Jun;148(4):387-95
16682964
J Biol Chem. 2003 Jun 6;278(23):20995-1002
12642577
Neuron. 2006 May 18;50(4):561-73
16701207
Biochem J. 2004 Mar 15;378(Pt 3):801-8
14656218
Nat Cell Biol. 2009 Apr;11(4):365-74
19337318
Proc Natl Acad Sci U S A. 2006 Jan 17;103(3):797-802
16407149
J Biol Chem. 2002 Nov 22;277(47):44925-31
12244098
J Biol Chem. 1999 Nov 19;274(47):33202-5
10559191
Proc Natl Acad Sci U S A. 2006 May 16;103(20):7877-82
16684876
Nature. 2002 Apr 25;416(6883):878-81
11976690
Proc Natl Acad Sci U S A. 2006 Jan 3;103(1):212-7
16371464
Trends Biochem Sci. 1999 Sep;24(9):340-1
10470031
J Biol Chem. 1992 Nov 25;267(33):24045-52
1358886
Endocrinology. 2008 Aug;149(8):3842-9
18450958
J Biol Chem. 2002 May 3;277(18):15897-903
11832491
Pharmacol Ther. 2007 Mar;113(3):488-506
17240454
Proc Natl Acad Sci U S A. 2008 Aug 26;105(34):12533-8
18719114
Nat Biotechnol. 1999 Sep;17(9):878-83
10471929
Neuron. 2004 Apr 22;42(2):269-81
15091342
J Biol Chem. 2002 Mar 1;277(9):6767-70
11756403
Nat Cell Biol. 2005 Dec;7(12):1179-90
16299499
J Biol Chem. 2003 Sep 12;278(37):34747-50
12881533
J Biol Chem. 2008 Dec 26;283(52):36698-710
18984596