Structure of Gα
            <sub>q</sub>
            -p63RhoGEF-RhoA Complex Reveals a Pathway for the Activation of RhoA by GPCRs

Lutz, Susanne; Shankaranarayanan, Aruna; Coco, Cassandra; Ridilla, Marc; Nance, M.R.; Vettel, Christiane; Baltus, Doris; Evelyn, Chris R.; Neubig, Richard R.; Wieland, Thomas; Tesmer, John J.G.

doi:10.1126/science.1147554

Cited by 207 publications

(266 citation statements)

References 28 publications

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“…Although a crystal packing effect can not be excluded, this rotation of the helical domain relative to the GTPase domain may represent a basic property of Gα q rather than a conformation induced by the binding of YM-254890. The configuration of the two domains of YM-254890-bound Gα i/q appears to overlap with the active form of Gα i/q (11,14) (Fig. S4B).…”

Section: Resultsmentioning

confidence: 99%

“…YM-254890 docks into the cleft between Linker 1 and Switch I (Linker 2), which connect the GTPase and helical domains. To our knowledge, this cleft has never been described as a critical contact site with other molecules, including Gα effectors (11,14), Gβγ (12, 13), GoLoco (15), GPCRs (16), or GPCR-mimic peptides (17). A structural comparison of our YM-254890-bound Gα i/ q βγ with the inhibitor-free heterotrimers containing Gα i1 or Gα t/i1 (12,13) suggested a slightly different configuration of the helical domain against the GTPase domain (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In Fig. S2, we used ectopically expressed Gα proteins in lysates from mammalian cells, because both Gα 14 and Gα 16 proteins were difficult to purify. The results of a previous study (5) and those of our studies indicate that YM-254890 selectively inhibits Gα q , Gα 11 , and Gα 14 among mammalian Gα members.…”

Section: Resultsmentioning

confidence: 99%

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Structural basis for the specific inhibition of heterotrimeric G _q protein by a small molecule

Nishimura

Kitano

Takasaki

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

233

317

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Heterotrimeric GTP-binding proteins (G proteins) transmit extracellular stimuli perceived by G protein-coupled receptors (GPCRs) to intracellular signaling cascades. Hundreds of GPCRs exist in humans and are the targets of a large percentage of the pharmaceutical drugs used today. Because G proteins are regulated by GPCRs, small molecules that directly modulate G proteins have the potential to become therapeutic agents. However, strategies to develop modulators have been hampered by a lack of structural knowledge of targeting sites for specific modulator binding. Here we present the mechanism of action of the cyclic depsipeptide YM-254890, which is a recently discovered G q -selective inhibitor. YM-254890 specifically inhibits the GDP/GTP exchange reaction of α subunit of G q protein (Gα q ) by inhibiting the GDP release from Gα q . X-ray crystal structure analysis of the Gα q βγ–YM-254890 complex shows that YM-254890 binds the hydrophobic cleft between two interdomain linkers connecting the GTPase and helical domains of the Gα q . The binding stabilizes an inactive GDP-bound form through direct interactions with switch I and impairs the linker flexibility. Our studies provide a novel targeting site for the development of small molecules that selectively inhibit each Gα subunit and an insight into the molecular mechanism of G protein activation.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Structural basis for the specific inhibition of heterotrimeric G _q protein by a small molecule

Nishimura

Kitano

Takasaki

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

233

317

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“…[41][42][43]. The ␣ q (wt) and ␣ q (R183C) cDNAs were a gift from B. R. Conklin and H. R. Bourne (University of California, San Francisco, CA).…”

Section: Results Not Shown) (Iii)mentioning

confidence: 99%

Differential Regulation of Phospholipase C-β2 Activity and Membrane Interaction by Gαq, Gβ1γ2, and Rac2

Gutman

Walliser

Piechulek

et al. 2010

Journal of Biological Chemistry

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We combined fluorescence recovery after photobleaching (FRAP) beam-size analysis with biochemical assays to investigate the mechanisms of membrane recruitment and activation of phospholipase C-␤ 2 (PLC␤ 2 ) by G protein ␣ q and ␤␥ dimers. We show that activation by ␣ q and ␤␥ differ from activation by Rac2 and from each other. Stimulation by ␣ q enhanced the plasma membrane association of PLC␤ 2 , but not of PLC␤ 2 ⌬, which lacks the ␣ q -interacting region. Although ␣ q resembled Rac2 in increasing the contribution of exchange to the FRAP of PLC␤ 2 and in enhancing its membrane association, the latter effect was weaker than with Rac2. Moreover, the membrane recruitment of PLC␤ 2 by ␣ q occurred by enhancing PLC␤ 2 association with fast-diffusing (lipid-like) membrane components, whereas stimulation by Rac2 led to interactions with slow diffusing membrane sites. On the other hand, activation by ␤␥ shifted the FRAP of PLC␤ 2 and PLC␤ 2 ⌬ to pure lateral diffusion 3-to 5-fold faster than lipids, suggesting surfing-like diffusion along the membrane. We propose that these different modes of PLC␤ 2 membrane recruitment may accommodate contrasting functional needs to hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ) in localized versus dispersed populations. PLC␤ 2 activation by Rac2, which leads to slow lateral diffusion and much faster exchange, recruits PLC␤ 2 to act locally on PtdInsP 2 at specific domains. Activation by ␣ q leads to lipid-like diffusion of PLC␤ 2 accompanied by exchange, enabling the sampling of larger, yet limited, areas prior to dissociation. Finally, activation by ␤␥ recruits PLC␤ 2 to the membrane by transient interactions, leading to fast "surfing" diffusion along the membrane, sampling large regions for dispersed PtdInsP 2 populations. Phospholipase C-␤ (PLC␤)4 isozymes hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ) to produce inositol 1,4,5-trisphosphate and diacylglycerol (1-3). They are activated to different extents by heterotrimeric G protein ␣ q subunits (␣ q ) and ␤␥ dimers (␤␥) (1-3). PLC␤ 2 is also activated by the Rho GTPases Rac and Cdc42 (4 -7). Activation by Rac has also been demonstrated for PLC␥ 2 (8). PLC␤ 2 has long been known to be expressed in hematopoietic cells (2, 3) but is also encountered in a variety of other cell types and tissues, including smooth muscle cells (9) and several brain regions (10, 11). Moreover, PLC␤ 2 was shown to be essential for taste perception via certain G protein-coupled oral taste receptors (12).Activation of PLC␤ 2 by ␣ q and related ␣ subunits requires the C-terminal region of the enzyme; mutants with deletions in this region (e.g. PLC␤ 2 ⌬, that lacks the Phe 819 -Glu 1166 segment) are resistant to stimulation by ␣ q , but undergo activation by ␤␥ and Rac/Cdc42 (7, 13-15). Recent results show that ␤␥ and Rac/Cdc42 activate PLC␤ 2 by interacting, at least in part, with different regions of the effector enzyme. Thus, although the pleckstrin homology (PH) domain of PLC␤ 2 is dispensable for activation of the enzyme by...

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“…CTNND1 is membrane-bound and plays a role in adherens junctions by regulating E-cadherin (41); it has also been shown to regulate WNT signaling (42). GNAQ is a cytoplasmic guaninine-nucleotide binding protein that signals to RhoA and is implicated in cell proliferation and migration (43). Low staining for CTNND1 (P = 0.023) was significantly associated with poor survival (Fig.…”

Section: Ccgs In Sb-driven Pancreatic Cancer Predict Human Pancreaticmentioning

confidence: 99%

Sleeping Beauty mutagenesis reveals cooperating mutations and pathways in pancreatic adenocarcinoma

Mann

Ward

Yew

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

207

219

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Pancreatic cancer is one of the most deadly cancers affecting the Western world. Because the disease is highly metastatic and difficult to diagnosis until late stages, the 5-y survival rate is around 5%. The identification of molecular cancer drivers is critical for furthering our understanding of the disease and development of improved diagnostic tools and therapeutics. We have conducted a mutagenic screen using Sleeping Beauty (SB) in mice to identify new candidate cancer genes in pancreatic cancer. By combining SB with an oncogenic Kras allele, we observed highly metastatic pancreatic adenocarcinomas. Using two independent statistical methods to identify loci commonly mutated by SB in these tumors, we identified 681 loci that comprise 543 candidate cancer genes (CCGs); 75 of these CCGs, including Mll3 and Ptk2, have known mutations in human pancreatic cancer. We identified point mutations in human pancreatic patient samples for another 11 CCGs, including Acvr2a and Map2k4. Importantly, 10% of the CCGs are involved in chromatin remodeling, including Arid4b, Kdm6a, and Nsd3, and all SB tumors have at least one mutated gene involved in this process; 20 CCGs, including Ctnnd1, Fbxo11, and Vgll4, are also significantly associated with poor patient survival. SB mutagenesis provides a rich resource of mutations in potential cancer drivers for cross-comparative analyses with ongoing sequencing efforts in human pancreatic adenocarcinoma. P ancreatic ductal adenocarcinoma is one of the most deadly forms of cancer. In the United States, the rate of mortality is nearly equal to the rate of new diagnoses (1). Early disease detection is rare, and of those patients diagnosed with early-stage disease, only 20% are candidates for surgical resection. Approximately 50% of patients develop highly metastatic disease, for which current treatment regimens provide little increase in longevity (2). Clearly, there is a need for better biomarkers of disease and the identification of new therapeutic targets, particularly for metastatic disease.Human pancreatic cancer develops from preinvasive neoplasias, typically intraepithelial neoplasias (PanINs), although intraductal papillary mucinous neoplasia and mucinous cystic neoplasia can also give rise to adenocarcinoma (3). The majority of pancreatic adenocarcinoma is of ductal origin and contains a large desmoplastic component thought to promote tumorigenesis by modulating the tumor microenvironment. Oncogenic KRAS mutations are found in 90% of human tumors, and they appear in early PanIN (4). The accumulation of additional inactivating driver mutations in P16/CDKN2A, TP53, and SMAD4 occurs with high frequency in later-stage PanIN, and these mutations are likely required for tumor progression to invasive adenocarcinoma.Recent high-throughput sequencing efforts have shown that the majority of somatic point mutations in primary pancreatic adenocarcinoma are missense mutations and that most occur at low frequency (5). Pancreatic cancers exhibit a very unstable genome, and through whole-genome...

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Structure of Gα _q -p63RhoGEF-RhoA Complex Reveals a Pathway for the Activation of RhoA by GPCRs

Cited by 207 publications

References 28 publications

Structural basis for the specific inhibition of heterotrimeric G _q protein by a small molecule

Structural basis for the specific inhibition of heterotrimeric G _q protein by a small molecule

Differential Regulation of Phospholipase C-β2 Activity and Membrane Interaction by Gαq, Gβ1γ2, and Rac2

Sleeping Beauty mutagenesis reveals cooperating mutations and pathways in pancreatic adenocarcinoma

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Structure of Gα q -p63RhoGEF-RhoA Complex Reveals a Pathway for the Activation of RhoA by GPCRs

Cited by 207 publications

References 28 publications

Structural basis for the specific inhibition of heterotrimeric G q protein by a small molecule

Structural basis for the specific inhibition of heterotrimeric G q protein by a small molecule

Differential Regulation of Phospholipase C-β2 Activity and Membrane Interaction by Gαq, Gβ1γ2, and Rac2

Sleeping Beauty mutagenesis reveals cooperating mutations and pathways in pancreatic adenocarcinoma

Contact Info

Product

Resources

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Structure of Gα _q -p63RhoGEF-RhoA Complex Reveals a Pathway for the Activation of RhoA by GPCRs

Structural basis for the specific inhibition of heterotrimeric G _q protein by a small molecule

Structural basis for the specific inhibition of heterotrimeric G _q protein by a small molecule