Biochemistry

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Competing Activities of Heterotrimeric G Proteins in Drosophila Wing Maturation

Drosophila genome encodes six alpha-subunits of heterotrimeric G proteins. The Gαs alpha-subunit is involved in the post-eclosion wing maturation, which consists of the epithelial-mesenchymal transition and cell death, accompanied by unfolding of the pupal wing into the firm adult flight organ. Here we show that another alpha-subunit Gαo can specifically antagonize the Gαs activities by competing for the Gβ13F/Gγ1 subunits of the heterotrimeric Gs protein complex. Loss of Gβ13F, Gγ1, or Gαs, but not any other G protein subunit, results in prevention of post-eclosion cell death and failure of the wing expansion. However, cell death prevention alone is not sufficient to induce the expansion defect, suggesting that the failure of epithelial-mesenchymal transition is key to the folded wing phenotypes. Overactivation of Gαs with cholera toxin mimics expression of constitutively activated Gαs and promotes wing blistering due to precocious cell death. In contrast, co-overexpression of Gβ13F and Gγ1 does not produce wing blistering, revealing the passive role of the Gβγ in the Gαs-mediated activation of apoptosis, but hinting at the possible function of Gβγ in the epithelial-mesenchymal transition. Our results provide a comprehensive functional analysis of the heterotrimeric G protein proteome in the late stages of Drosophila wing development

Drosophila GoLoco-Protein Pins is a target of G{alpha}o-mediated G Protein-coupled Receptor Signaling

G protein-coupled receptors (GPCRs) transduce their signals through trimeric G proteins, inducing guanine nucleotide exchange on their Galpha-subunits; the resulting Galpha-GTP transmits the signal further inside the cell. GoLoco domains present in many proteins play important roles in multiple trimeric G protein-dependent activities, physically binding Galpha-subunits of the Galphai/o class. In most cases GoLoco binds exclusively to the GDP-loaded form of the Galpha-subunits. Here we demonstrate that the poly-GoLoco-containing protein Pins of Drosophila can bind to both GDP- and GTP-forms of Drosophila Galphao. We identify Pins GoLoco domain 1 as necessary and sufficient for this unusual interaction with Galphao-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 Galphao-mediated GPCR receptor signaling, e.g., in the context of the nervous system development, where Galphao acts downstream from Frizzled and redundantly with Galphai to control the asymmetry of cell divisions

Prevention of apoptosis is associated with, but is not sufficient to induce, the failure of wing expansion.

<p><b>A–B</b>. Wild-type wings are fully expanded and show GFP (A) or F-actin (B) staining only on the margin and along the veins, demonstrating that the adult wings are mostly dead structures. <b>C–G</b>. Downregulation of the Gs pathway by overexpression of Gαo (C, D) or by expression of RNAi constructs targeting Gβ13F (E), Gγ1 (F), or Gαs (G) leads to both failure of wing expansion and prevention of apoptosis, as visualized by persistence of F-actin- (D) and GFP-positive cells (C, E–G). <b>H–J</b>. DAPI nuclear staining. Overexpression of Gαo in aged wings leads to the DAPI staining pattern (J) characteristic of the young (ca. 1h-old, H) wild-type wings; aged wild-type wing only shows DAPI staining along the veins (I). <b>K</b>. Expression of the apoptosis inhibitor p35 prevents cell death throughout the wing as seen by persistence of GFP-positive cells, but does not cause the failure of wing expansion. All wings presented here are from <i>MS1096-Gal4; UAS-GFP</i> flies which are ≥1 day-old (except for the wing of panel (H)).</p

Proteomic analysis identifies a few <i>Drosophila</i> proteins specifically interacting with the GDP- or the GTP-loaded forms of Gαo.

<p><b>A</b>. SDS-PAGE of the <i>Drosophila</i> head proteins retained by the GDP- or the GTPγS-loaded CNBr-immobilized Gαo. The single band enriched in the GDP-lane is indicated by the green arrow. The two bands enriched in the GTPγS-lane are indicated by the blue and magenta arrows. Positions of molecular weight markers are shown to the right of the gel. <b>B–C</b>. 2D-gel of the same samples as shown in (A), DIGE-labeled and loaded on the same gel, visualized in the Cy3-channel (B, Gαo-GDP-interacting proteins) and in the Cy5-channel (C, Gαo-GTPγS-interacting proteins). The spots enriched in one or the other samples are outlined in green, blue and magenta. <b>D–F</b>. High magnification of the spots enriched in the Gαo-GDP- <i>vs</i> the Gαo- GTPγS -interacting proteins, together with the quantification of the normalized intensity of these spots between the two samples. Quantification in (E, F) is presented as the sum of intensities of all the spots outlined (five in (E), two in (F)). The spot in (D) was identified as Gβ13F, spots 1 and 4 (from left to right) in (E) were identified as β1-Tubulin, spot 2 in (F) was identified as Hsc70-3; other spots failed to be identified by LC-MSMS.</p

Downregulation of Gγ1 or Gβ13F, but not of any other Gβ or Gγ subunit, leads to the failure of wing expansion.

<p>Representative wings expressing the RNAi constructs targeting Gγ30A (B), Gγ1 (C), Gβ5 (D), Gβ76C (E), or Gβ13F (F) are shown along with the <i>MS1096-Gal4</i> driver line alone (A).</p

The list of <i>Drosophila</i> Gα, Gβ, and Gγ subunits, with the information on their function and human homologies.

<p>The list of <i>Drosophila</i> Gα, Gβ, and Gγ subunits, with the information on their function and human homologies.</p

Overactivation of Gαs, but not Gβγ, leads to wing blistering due to precocious apoptosis.

<p><b>A</b>. Expression of the constitutively active form of Gαs by multiple <i>Gal4</i> drivers produces the characteristic wing blistering. The picture shown represents an <i>OK10-Gal4/UAS-Gαs[GTP]</i> fly. <b>B</b>. Activation of the endogenous Gαs by expression of cholera toxin with multiple drivers also produces wing blistering (arrows). The picture shown represents a <i>Vg-Gal4, UAS-flp; UAS>w⁺>cholera toxin</i> fly. <b>C–E</b>. Expression of Gβ13F alone (C), Gγ1 alone (D), or both (E) by multiple drivers including <i>OK10-Gal4</i> never produces the wing blistering. The pictures shown represent wings of <i>MS1096-Gal4; UAS-Gβ/γ</i> flies. <b>F</b>. Sequestration of Gβγ by Gαo does not prevent wing blistering induced by the constitutively active form of Gαs. The picture shown represents an <i>MS1096-Gal4, UAS-Gαo</i>; <i>OK10-Gal4/UAS-Gαs[GTP]</i> fly.</p

A model of the action of components of the heterotrimeric Gs complex in wing maturation.

<p>The neurohormone bursicon acts on the Gs-coupled GPCR <i>rickets</i> expressed in wing cells. The GPCR activity leads to dissociation of the heterotrimeric Gs complex into GTP-loaded Gαs and free Gβγ-heterodimer. Gαs[GTP] activates the cAMP-PKA pathway to promote apoptosis. Gβγ, on the other hand, acts to induce the epithelial-mesenchymal transition. These two processes, acting in coordination, lead to post-eclosion wing expansion and solidification. Expression of the constitutively active Gαs or cholera toxin stimulates the Gαs-dependent branch in this signaling. Expression of Gαo inhibits this signaling through sequestration of the Gβγ-subunits.</p

Downregulation of Gαs, but not other Gα-subunits, leads to the failure of wing expansion.

<p>Representative wings expressing the RNAi constructs targeting Gαi (B), Gαq (C), Gαf (D), or Gαs (E) are shown along with the <i>MS1096-Gal4</i> driver line alone (A) and the wing of the concertina homozygous mutant fly (<i>cta^−/−</i>, F).</p