Engineering GPCR signaling pathways with RASSLs

Conklin, Bruce R.; Hsiao, Edward C.; Claeysen, Sylvie; Dumuis, Aline; Srinivasan, Supriya; Forsayeth, John; Guettier, Jean Marc; Chang, Wei Chun; Pei, Ying; McCarthy, Ken D.; Nissenson, Robert A.; Wess, Jürgen; Bockaert, Joël; Roth, Bryan L.

doi:10.1038/nmeth.1232

Cited by 231 publications

(205 citation statements)

References 37 publications

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“…The use of engineered GPCRs with modified ligand binding properties to study various aspects of GPCR biology has been pioneered by Conklin and coworkers (20). However, in contrast to previous studies, the present in vivo study uses engineered GPCRs that are unable to bind endogenous ligand but can be efficiently activated by an otherwise pharmacologically inert drug.…”

Section: Discussionmentioning

confidence: 99%

A chemical-genetic approach to study G protein regulation of β cell function in vivo

Guettier

Gautam

Scarselli

et al. 2009

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

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312

382

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Impaired functioning of pancreatic ␤ cells is a key hallmark of type 2 diabetes. ␤ cell function is modulated by the actions of different classes of heterotrimeric G proteins. The functional consequences of activating specific ␤ cell G protein signaling pathways in vivo are not well understood at present, primarily due to the fact that ␤ cell G protein-coupled receptors (GPCRs) are also expressed by many other tissues. To circumvent these difficulties, we developed a chemicalgenetic approach that allows for the conditional and selective activation of specific ␤ cell G proteins in intact animals. Specifically, we created two lines of transgenic mice each of which expressed a specific designer GPCR in ␤ cells only. Importantly, the two designer receptors differed in their G protein-coupling properties (Gq/11 versus Gs). They were unable to bind endogenous ligand(s), but could be efficiently activated by an otherwise pharmacologically inert compound (clozapine-N-oxide), leading to the conditional activation of either ␤ cell Gq/11 or Gs G proteins. Here we report the findings that conditional and selective activation of ␤ cell Gq/11 signaling in vivo leads to striking increases in both first-and second-phase insulin release, greatly improved glucose tolerance in obese, insulin-resistant mice, and elevated ␤ cell mass, associated with pathway-specific alterations in islet gene expression levels. Selective stimulation of ␤ cell Gs triggered qualitatively similar in vivo metabolic effects. Thus, this developed chemical-genetic strategy represents a powerful approach to study G protein regulation of ␤ cell function in vivo.beta cells ͉ G protein-coupled receptors ͉ transgenic mice ͉ type 2 diabetes T ype 2 diabetes has emerged as one of the major threats to human health in the 21st century (1). Impaired function of pancreatic ␤ cells is one of the key hallmarks of type 2 diabetes, and therapies targeted at improving ␤ cell function are predicted to offer considerable therapeutic benefit (2).␤ Cell function is modulated by the actions of different classes of heterotrimeric G proteins which are the immediate downstream targets of a multitude of G protein-coupled receptors (GPCRs). Like most other cell types, pancreatic ␤ cells are predicted to express many different GPCRs (3-5). Several lines of evidence suggest that activation of G s -coupled receptors expressed by pancreatic ␤ cells, including the glucagon-like peptide (GLP-1) receptor, improves ␤ cell function and can increase in ␤ cell mass via cAMP-dependent mechanisms (5-7). Pancreatic ␤ cells also express several G q/11 -coupled receptors, including the M 3 muscarinic acetylcholine (ACh) receptor (M3R) and GPR40, which can promote insulin release in an agonist-dependent fashion [for recent reviews, see (5,8)].Studies with GLP-1 receptor agonists have yielded detailed information about the beneficial effects of G s signaling on ␤ cell function and whole body glucose homeostasis (note that the GLP-1 receptor is enriched in pancreatic ␤ cells) (5-7). In contrast, much...

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Section: Discussionmentioning

confidence: 99%

A chemical-genetic approach to study G protein regulation of β cell function in vivo

Guettier

Gautam

Scarselli

et al. 2009

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

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312

382

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“…We conceived of an approach to direct cellular homing to small molecules by expressing, in motile cells, engineered G proteincoupled receptors (GPCRs) called receptors activated solely by a synthetic ligand (RASSLs) (20,21).…”

mentioning

confidence: 99%

Synthetic control of mammalian-cell motility by engineering chemotaxis to an orthogonal bioinert chemical signal

Park

Rhau

Hermann

et al. 2014

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

104

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Directed migration of diverse cell types plays a critical role in biological processes ranging from development and morphogenesis to immune response, wound healing, and regeneration. However, techniques to direct, manipulate, and study cell migration in vitro and in vivo in a specific and facile manner are currently limited. We conceived of a strategy to achieve direct control over cell migration to arbitrary user-defined locations, independent of native chemotaxis receptors. Here, we show that genetic modification of cells with an engineered G protein-coupled receptor allows us to redirect their migration to a bioinert drug-like small molecule, clozapine-Noxide (CNO). The engineered receptor and small-molecule ligand form an orthogonal pair: The receptor does not respond to native ligands, and the inert drug does not bind to native cells. CNOresponsive migration can be engineered into a variety of cell types, including neutrophils, T lymphocytes, keratinocytes, and endothelial cells. The engineered cells migrate up a gradient of the drug CNO and transmigrate through endothelial monolayers. Finally, we demonstrate that T lymphocytes modified with the engineered receptor can specifically migrate in vivo to CNO-releasing beads implanted in a live mouse. This technology provides a generalizable genetic tool to systematically perturb and control cell migration both in vitro and in vivo. In the future, this type of migration control could be a valuable module for engineering therapeutic cellular devices.GPCR | cellular therapeutics | synthetic biology

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“…45 G-protein coupled receptors (GPCRs) are seven-transmembrane proteins that function as signaling switches throughout the body and regulate virtually every physiological response. 46,47 This is one of the largest gene families targeted for drug discovery. 47 Peptides (usually around 20 amino acids in length) that mimic GPCR-transmembrane domains [48][49][50] are thought to inhibit GPCR oligomerization.…”

mentioning

confidence: 99%

Peptidic modulators of protein‐protein interactions: Progress and challenges in computational design

Rubinstein

Niv

2009

Biopolymers

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With the decline in productivity of drug‐development efforts, novel approaches to rational drug design are being introduced and developed. Naturally occurring and synthetic peptides are emerging as novel promising compounds that can specifically and efficiently modulate signaling pathways in vitro and in vivo. We describe sequence‐based approaches that use peptides to mimic proteins in order to inhibit the interaction of the mimicked protein with its partners. We then discuss a structure‐based approach, in which protein‐peptide complex structures are used to rationally design and optimize peptidic inhibitors. We survey flexible peptide docking techniques and discuss current challenges and future directions in the rational design of peptidic inhibitors. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 505–513, 2009.This article was originally published online as an accepted preprint. The “Published Online”date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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Engineering GPCR signaling pathways with RASSLs

Cited by 231 publications

References 37 publications

A chemical-genetic approach to study G protein regulation of β cell function in vivo

A chemical-genetic approach to study G protein regulation of β cell function in vivo

Synthetic control of mammalian-cell motility by engineering chemotaxis to an orthogonal bioinert chemical signal

Peptidic modulators of protein‐protein interactions: Progress and challenges in computational design

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