The alpha subunit of the G protein G13 regulates activity of one or more Gli transcription factors independently of smoothened.
ABSTRACT Smoothened (Smo) is a seven-transmembrane (7-TM) receptor that is essential to most actions of the Hedgehog family of morphogens. We found previously that Smo couples to members of the G(i) family of heterotrimeric G proteins, which in some cases are integral although alone insufficient in the activation of Gli transcription factors through Hedgehog signaling. In response to a report that the G(12/13) family is relevant to Hedgehog signaling as well, we re-evaluated the coupling of Smo to one member of this family, G(13), and investigated the capacity of this and other G proteins to activate one or more of forms of Gli. We found no evidence that Smo couples directly to G(13). We found nonetheless that Gα(13) and to some extent Gα(q) and Gα(12) are able to effect activation of Gli(s). This capacity is realized in some cells, e.g. C3H10T1/2, MC3T3, and pancreatic cancer cells, but not all cells. The mechanism employed is distinct from that achieved through canonical Hedgehog signaling, as the activation does not involve autocrine signaling or in any other way require active Smo and does not necessarily involve enhanced transcription of Gli1. The activation by Gα(13) can be replicated through a G(q)/G(12/13)-coupled receptor, CCK(A), and is attenuated by inhibitors of p38 mitogen-activated protein kinase and Tec tyrosine kinases. We posit that G proteins, and perhaps G(13) in particular, provide access to Gli that is independent of Smo and that they thus establish a basis for control of at least some forms of Gli-mediated transcription apart from Hedgehogs.
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ABSTRACT: Smoothened (Smo) is a 7-transmembrane (7-TM) protein essential to the activation of Gli transcription factors (Gli) by Hedgehog morphogens. The structure of Smo implies interactions with heterotrimeric G proteins, yet the degree to which G proteins participate in the actions of Hedgehogs remains controversial. We posit that the G(i) family of G proteins provides to Hedgehogs the ability to expand well beyond the bounds of Gli. In this regard, we evaluate here the efficacy of Smo as it relates to the activation of G(i). We do so by comparing Smo to the 5-hydroxytryptamine(1A) (5-HT(1A)) receptor, a quintessential G(i)-coupled receptor. We find using [(35)S]guanosine 5'-(3-O-thio)triphosphate, first with forms of G(i) endogenous to HEK-293 cells made to express epitope-tagged receptors and second with individual forms of Gα(i) fused to the C terminus of each receptor, that Smo is equivalent to the 5-HT(1A) receptor in the assay as it relates to capacity to activate G(i). This finding is true regardless of subtype of G(i) (e.g. G(i2), G(o), and G(z)) tested. We also demonstrate that Smo endogenous to HEK-293 cells, ostensibly through inhibition of adenylyl cyclase, decreases intracellular levels of cAMP. The equivalence of Smo to the 5-HT(1A) receptor in the activation of G(i) and the decrease through endogenous Smo of cAMP speak to Smo as a receptor that can engage not just Gli but any of a variety of more immediate effectors.Molecular pharmacology 01/2013; · 4.53 Impact Factor
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ABSTRACT: Small cell lung carcinoma (SCLC) often features the upregulation of the Sonic hedgehog (Shh) pathway leading to activation of Gli transcription factors. SCLC cells secrete bombesin (BBS)-like neuropeptides that act as autocrine growth factors. Here, we show that SCLC tumor samples feature co-expression of Shh and BBS-cognate receptor (gastrin-releasing peptide receptor (GRPR)). We also demonstrate that BBS activates Gli in SCLC cells, which is crucial for BBS-mediated SCLC proliferation, because cyclopamine, an inhibitor of the Shh pathway, hampered the BBS-mediated effects. BBS binding to GRPR stimulated Gli through its downstream Gαq and Gα12/13 GTPases, and consistently, other Gαq and Gα13 coupled receptors (such as muscarinic receptor, m1, and thrombin receptor, PAR-1) and constitutively active GαqQL and Gα12/13QL mutants stimulated Gli. By using cells null for Gαq and Gα12/13, we demonstrate that these G proteins are strictly necessary for Gli activation by BBS. Moreover, by using constitutively active Rho small G-protein (Rho QL) as well as its inhibitor, C3 toxin, we show that Rho mediates G-protein-coupled receptor (GPCR)-, Gαq- and Gα12/13-dependent Gli stimulation. At the molecular level, BBS caused a significant increase in Shh gene transcription and protein secretion that was dependent on BBS-induced GPCR/Gαq-12/13/Rho mediated activation of nuclear factor κB (NFκB), which can stimulate a NF-κB response element in the Shh gene promoter. Our data identify a novel molecular network acting in SCLC linking autocrine BBS and Shh circuitries and suggest Shh inhibitors as novel therapeutic strategies against this aggressive cancer type.Oncogene advance online publication, 21 April 2014; doi:10.1038/onc.2014.104.Oncogene 04/2014; · 8.56 Impact Factor
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ABSTRACT: Hedgehog (Hh) signaling plays fundamental roles in morphogenesis, tissue repair, and human disease. Initiation of Hh signaling is controlled by the interaction of two multipass membrane proteins, patched (Ptc) and smoothened (Smo). Recent studies identify Smo as a G-protein coupled receptor (GPCR)-like protein that signals through large G-protein complexes which contain the Gαi subunit. We hypothesize Regulator of G-Protein Signaling (RGS) proteins, and specifically RGS5, are endogenous repressors of Hh signaling via their ability to act as GTPase activating proteins (GAPs) for GTP-bound Gαi, downstream of Smo. In support of this hypothesis, we demonstrate that RGS5 over-expression inhibits sonic hedgehog (Shh)-mediated signaling and osteogenesis in C3H10T1/2 cells. Conversely, signaling is potentiated by siRNA-mediated knock-down of RGS5 expression, but not RGS4 expression. Furthermore, using immuohistochemical analysis and co-immunoprecipitation (Co-IP), we demonstrate that RGS5 is present with Smo in primary cilia. This organelle is required for canonical Hh signaling in mammalian cells, and RGS5 is found in a physical complex with Smo in these cells. We therefore conclude that RGS5 is an endogenous regulator of Hh-mediated signaling and that RGS proteins are potential targets for novel therapeutics in Hh-mediated diseases.PLoS ONE 01/2013; 8(4):e61421. · 3.53 Impact Factor