DrosophilaGoLoco-Protein Pins Is a Target of Gα_o-mediated G Protein–coupled Receptor Signaling

Abstract

Drosophila G␣ o . We identify Pins GoLoco domain 1 as necessary and sufficient for this unusual interaction with G␣ o -GTP. We further pinpoint a lysine residue located centrally in this domain as necessary for the interaction. Our studies thus identify Drosophila Pins as a target of G␣ o -mediated GPCR receptor signaling, e.g., in the context of the nervous system development, where G␣ o acts downstream from Frizzled and redundantly with G␣ i to control the asymmetry of cell divisions. INTRODUCTIONTrimeric G proteins transduce the signals from G proteincoupled receptors (GPCRs), the largest receptor family in the animal kingdom (Pierce et al., 2002). Signal specificity is mainly represented by the ␣-subunits of the trimeric G proteins; 16 genes for the ␣-subunits are present in the human genome, and six in Drosophila (Malbon, 2005). Both in flies and mammals, G␣ o is the predominant G␣-subunit in the nervous system (Sternweis and Robishaw, 1984;Wolfgang et al., 1990); up to 10% of the whole plasma membrane proteins of the neuronal growth cones is represented by the trimeric G o protein (Strittmatter et al., 1990). G␣ o is required for the proper brain functioning and development (Jiang et al., 1998;Ferris et al., 2006), e.g., controlling neurite outgrowth (Bromberg et al., 2008). Among the brain GPCRs activating G␣ o are the dopamine, serotonin, adenosine, cannabinoid, glutamate, and other receptors (Offermanns, 2003;Bromberg et al., 2008). Additional developmental functions of G␣ o are the transduction of the evolutionary conserved Frizzled receptors (Egger-Adam and Katanaev, 2008) and the regulation of the heart development and physiology (Valenzuela et al., 1997;Fremion et al., 1999).In the resting state the trimeric G proteins exist as complexes of the GDP-bound ␣-subunit and the ␤-and ␥-subunits. On ligand activation, GPCRs serve as guanine nucleotide exchange factors, catalyzing the substitution of GDP for GTP on the G␣-subunit. This leads to dissociation of the complex into the GTP-loaded G␣ and the ␤␥-heterodimer. Both components of the initial complex can interact with downstream effectors (Gilman, 1987).GoLoco domains (Willard et al., 2004) present in many different proteins across the animal kingdom can specifically bind ␣-subunits of the G i/o class of trimeric G protein (G␣ i , G␣ o , G␣ t , and G␣ z ) and thus might serve as a hallmark of a subclass of G␣ i/o target proteins. For example, interaction of G␣ i/o with the GoLoco-containing protein Rap1Gap (a negative regulator of a small G protein Rap1) has been proposed as a mechanism of GPCR-induced neurite outgrowth (Jordan et al., 1999;Jordan et al., 2005). However, in the majority of cases GoLoco domains bind to the GDP-, and not the GTP-loaded forms of free G␣-subunits (Willard et al., 2004); furthermore, some GoLoco motifs are able to dissociate the trimeric G protein complexes without nucleotide exchange (Takesono et al., 1999;Ghosh et al., 2003). These observations have led to proposition that GoLoco-containing proteins may serve not as targ...

Regulation of the AGS3·Gαi Signaling Complex by a Seven-transmembrane Span Receptor*

“…However, the mechanisms controlling the interactions between GPR-containing proteins and their G-protein partners remain poorly understood. Such signals may involve coordinated positioning of GPR proteins within the cell, receptor-activated signaling cascades, regulatory proteins binding to non-GPR domains of the proteins, and/or non-receptor guanine nucleotide exchange factors acting on a GPR⅐G␣ i complex (2,3,(25)(26)(27)(28)(29)(30)(31)(32).…”

AGS-3 AltersCaenorhabditis elegansBehavior after Food Deprivation via RIC-8 Activation of the Neural G Protein Gαo

“…Previous in vitro studies of the interactions between Gα i/o proteins and AGS3 family proteins were consistent with either the AGS3 protein inactivating Gα signaling by acting as a guanine nucleotide dissociation inhibitor, or activating signaling by either Gα or Gβγ (Natochin et al, 2000; Bernard et al, 2001; Kopein and Katanaev, 2009). Our work clearly shows that AGS-3 acts upstream of Gα o to activate signaling via the Gα subunit.…”

“…We found that full-length GST-AGS-3 bound to only Gα o -GDP, the inactive state of the G protein, while the C-terminal fragment of AGS-3 containing the four GPR domains could additionally bind Gα o -GTP, the activated state. Thus, the Gα binding activity of full-length AGS-3 is similar to that of mammalian AGS3 and LGN and Drosophila Pins (Mochizuki et al, 1996; Takesono et al, 1999; Schaefer et al, 2000; Bernard et al, 2001; Kopein and Katanaev, 2009). The C-terminal GPR fragment of Pins also has the Gα-GTP binding activity we saw for the GPR fragment of AGS-3 (Kopein and Katanaev, 2009).…”

“…Thus, the Gα binding activity of full-length AGS-3 is similar to that of mammalian AGS3 and LGN and Drosophila Pins (Mochizuki et al, 1996; Takesono et al, 1999; Schaefer et al, 2000; Bernard et al, 2001; Kopein and Katanaev, 2009). The C-terminal GPR fragment of Pins also has the Gα-GTP binding activity we saw for the GPR fragment of AGS-3 (Kopein and Katanaev, 2009). In PINS, this activity is masked in the full-length protein by an interaction between the TPR and GPR repeats.…”

“…In vitro, GPR domains bind to the inactive GDP-bound state of Gα i/o , inhibit nucleotide exchange by Gα, and compete with the Gβγ dimer for Gα binding (Takesono et al, 1999; Natochin et al, 2000; Bernard et al, 2001; Kopein and Katanaev, 2009). Additional biochemical studies have shown that the guanine nucleotide exchange factor Ric-8A can activate Gα by promoting exchange of GDP for GTP when Gα is bound to a GPR domain (Tall and Gilman, 2005; Thomas et al, 2008), but not when Gα is bound to Gβγ (Tall et al, 2003).…”