G Protein–Coupled Receptor–Mediated Activation of p110β by Gβγ Is Required for Cellular Transformation and Invasiveness

Dbouk, Hashem A.; Vadas, Oscar; Shymanets, Aliaksei; Burke, John E.; Salamon, Rachel S.; Khalil, Bassem D.; Barrett, Mathew O.; Waldo, Gary L.; Surve, Chinmay R.; Hsueh, Christine; Perišić, Olga; Harteneck, Christian; Shepherd, Peter R.; Harden, T. Kendall; Smrcka, Alan V.; Taussig, Ronald; Bresnick, Anne R.; Nürnberg, Bernd; Williams, Roger L.; Backer, Jonathan M.

doi:10.1126/scisignal.2003264

Cited by 138 publications

(229 citation statements)

References 47 publications

(72 reference statements)

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“…Gbg residues 23-27 are used for regulation of PLCb , and for interaction with AGS8 when the hotspot is otherwise occupied (Yuan et al, 2007). It has also been suggested that PI3K interacts with the Gb NT at residues 31-45 (Dbouk et al, 2012). However, Gb NT residues 23-27 are also critical for AC6 regulation by Gbg and thus cannot solely explain the differential sensitivity to SIGK.…”

Section: Discussionmentioning

confidence: 97%

Adenylyl Cyclase 5 Regulation by GβγInvolves Isoform-Specific Use of Multiple Interaction Sites

et al. 2015

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Adenylyl cyclase (AC) converts ATP into cyclic AMP (cAMP), an important second messenger in cell signaling. Heterotrimeric G proteins and other regulators are important for control of AC activity. Depending on the AC isoform, Gbg subunits can either conditionally stimulate or inhibit cAMP synthesis. We previously showed that the Ga s -bg heterotrimer binds to the N terminus (NT) of type 5 AC (AC5). We now show that Gbg binds to the NT of a wide variety of AC isoforms. We hypothesized that Gbg/AC5 interactions involving inactive heterotrimer and Gbg stimulation of AC5 were separable events. Mutations of the Gbg "hotspot" show that this site is necessary for AC5 stimulation but not for interactions with the first 198 aa of AC5NT, which is a G protein scaffolding site. This contrasts with AC6, where the Gbg hotspot is required for both interactions with AC6NT and for stimulation of AC6. Additionally, the SIGK hotspot peptide disrupts Gbg regulation of AC isoforms 1, 2, and 6, but not AC5. Gbg also binds the C1/C2 catalytic domains of AC5 and AC6. Finally, cellular interactions with full-length AC5 depend on multiple sites on Gbg. This suggests an isoform-specific mechanism in which bound Gbg at the AC5NT is ideally situated for spatiotemporal control of AC5. We propose Gbg regulation of AC involves multiple binding events, and the role of the AC NT for mechanisms of regulation by heterotrimeric G protein subunits is isoform-specific.

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Section: Discussionmentioning

confidence: 97%

Adenylyl Cyclase 5 Regulation by GβγInvolves Isoform-Specific Use of Multiple Interaction Sites

et al. 2015

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“…Although there is a crystal structure of p110γ (30), no structural information or even domain organization for the p101 regulatory subunit is known, and similarly, there is no structural information about PI3Kγ interaction with membranes. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a useful tool for analyzing interactions of proteins with membranes (31), and it has been important for understanding how PI3K complexes become activated on lipid membranes (24). The method also provides insight into protein dynamics that is difficult or impossible to obtain with other tools (32)(33)(34)(35).…”

Section: Significancementioning

confidence: 99%

“…In contrast to other class I PI3Ks, p110γ uniquely associates with a p101 or a p87 (also called p84) regulatory subunit (20)(21)(22)(23). PI3Kγ shares with the class I A PI3K p110β the ability to be directly activated by Gβγ heterodimers (24)(25)(26). The p110γ catalytic subunit on its own can be activated only to a limited extent by Gβγ heterodimers, whereas maximal activation requires association with the p101 subunit and with Ras (21,25,27,28).…”

mentioning

confidence: 99%

Molecular determinants of PI3Kγ-mediated activation downstream of G-protein–coupled receptors (GPCRs)

Vadas

Dbouk²,

Shymanets

et al. 2013

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

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Phosphoinositide 3-kinase gamma (PI3Kγ) has profound roles downstream of G-protein-coupled receptors in inflammation, cardiac function, and tumor progression. To gain insight into how the enzyme's activity is shaped by association with its p101 adaptor subunit, lipid membranes, and Gβγ heterodimers, we mapped these regulatory interactions using hydrogen-deuterium exchange mass spectrometry. We identify residues in both the p110γ and p101 subunits that contribute critical interactions with Gβγ heterodimers, leading to PI3Kγ activation. Mutating Gβγ-interaction sites of either p110γ or p101 ablates G-protein-coupled receptor-mediated signaling to p110γ/p101 in cells and severely affects chemotaxis and cell transformation induced by PI3Kγ overexpression. Hydrogen-deuterium exchange mass spectrometry shows that association with the p101 regulatory subunit causes substantial protection of the RBD-C2 linker as well as the helical domain of p110γ. Lipid interaction massively exposes that same helical site, which is then stabilized by Gβγ. Membrane-elicited conformational change of the helical domain could help prepare the enzyme for Gβγ binding. Our studies and others identify the helical domain of the class I PI3Ks as a hub for diverse regulatory interactions that include the p101, p87 (also known as p84), and p85 adaptor subunits; Rab5 and Gβγ heterodimers; and the β-adrenergic receptor kinase.

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“…In contrast, the p110b RBD interacts with Rab5 GTPase (Christoforidis et al, 1999) and Rho GTPase family members (Fritsch et al, 2013), and specifically, Rac potentiates p110b GPCR responses (Guillermet-Guibert et al, 2008;Dbouk et al, 2012;Fritsch et al, 2013). The class II PI3K (PI3KC2) subfamily has three members in vertebrates (see poster), only one member in worm and fly, and also none in yeast (Brown and Auger, 2011;Falasca and Maffucci, 2012).…”

Section: Pi3k Protein Domains and Regulatory Subunitsmentioning

confidence: 99%

Classes of phosphoinositide 3-kinases at a glance

Jean

Kiger

2014

Journal of Cell Science

298

247

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The phosphoinositide 3-kinase (PI3K) family is important to nearly all aspects of cell and tissue biology and central to human cancer, diabetes and aging. PI3Ks are spatially regulated and multifunctional, and together, act at nearly all membranes in the cell to regulate a wide range of signaling, membrane trafficking and metabolic processes. There is a broadening recognition of the importance of distinct roles for each of the three different PI3K classes (I, II and III), as well as for the different isoforms within each class. Ongoing issues include the need for a better understanding of the in vivo complexity of PI3K regulation and cellular functions. This Cell Science at a Glance article and the accompanying poster summarize the biochemical activities, cellular roles and functional requirements for the three classes of PI3Ks. In doing so, we aim to provide an overview of the parallels, the key differences and crucial interplays between the regulation and roles of the three PI3K classes.

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G Protein–Coupled Receptor–Mediated Activation of p110β by Gβγ Is Required for Cellular Transformation and Invasiveness

Cited by 138 publications

References 47 publications

Adenylyl Cyclase 5 Regulation by GβγInvolves Isoform-Specific Use of Multiple Interaction Sites

Adenylyl Cyclase 5 Regulation by GβγInvolves Isoform-Specific Use of Multiple Interaction Sites

Molecular determinants of PI3Kγ-mediated activation downstream of G-protein–coupled receptors (GPCRs)

Classes of phosphoinositide 3-kinases at a glance

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