RESEARCH ARTICLE
Chemical Genetics Reveals an RGS/G-Protein Role in the Action of a Compound
Kevin Fitzgerald1, Svetlana Tertyshnikova1, Lisa Moore2, Lynn Bjerke2, Ben Burley2, Jian Cao1, Pamela Carroll1, Robert Choy2, Steve Doberstein2, Yves Dubaquie1, Yvonne Franke2, Jenny Kopczynski2, Hendrik Korswagen3, Stanley R. Krystek Jr.1, Nicholas J. Lodge1, Ronald Plasterk3, John Starrett1, Terry Stouch1, George Thalody1, Honey Wayne2, Alexander van der Linden3, Yongmei Zhang1, Stephen G. Walker1, Mark Cockett1, Judi Wardwell-Swanson1, Petra Ross-Macdonald1*, Rachel M. Kindt2
1 Bristol-Myers Squibb Pharmaceutical Research Institute, Pennington, New Jersey, United States of America, 2 Exelixis Incorporated, South San Francisco, California, United States of America, 3 Hubrecht Laboratory, Centre for Biomedical Genetics, Utrecht, Netherlands * To whom correspondence should be addressed. E-mail: Petra.RossMacdonald@bms.com
Funding. This research was funded by Bristol-Myers Squibb and Exelixis Incorporated.
Competing interests. This research was funded by Bristol-Myers Squibb and Exelixis Incorporated.
Editor: Susan Mango, Huntsman Cancer Institute, United States of America
We report here on a chemical genetic screen designed to address the mechanism of action of a small molecule. Small molecules that were active in models of urinary incontinence were tested on the nematode Caenorhabditis elegans, and the resulting phenotypes were used as readouts in a genetic screen to identify possible molecular targets. The mutations giving resistance to compound were found to affect members of the RGS protein/G-protein complex. Studies in mammalian systems confirmed that the small molecules inhibit muscarinic G-protein coupled receptor (GPCR) signaling involving G-αq (G-protein alpha subunit). Our studies suggest that the small molecules act at the level of the RGS/G-αq signaling complex, and define new mutations in both RGS and G-αq, including a unique hypo-adapation allele of G-αq. These findings suggest that therapeutics targeted to downstream components of GPCR signaling may be effective for treatment of diseases involving inappropriate receptor activation.
PLoS Genet 2(4): e57. This is an open-access article distributed under the terms of the Creative Commons Attribution License.
Synopsis
The authors have utilized Caenorhabditis elegans, and yeast genetics, combined with mammalian tissue and cell culture experiments to investigate the mechanism of action of a unique set of small molecules. These molecules are active in tissue models of urinary incontinence and allow for increased bladder filling. In the course of studying sensitivity and resistance to these compounds, Fitzgerald et al. uncovered novel alleles of RGS and Gq proteins. Further characterization of one such allele identified that its action conferred a hypo-adaptive phenotype on yeast during pheromone signaling assays. Their data as a whole indicate that these small molecules are able to diminish signaling from G-protein coupled receptors (GPCR) downstream of the receptors themselves. Since GPCR signaling is very important in many diseases in humans, the novel mechanism of these compounds may offer new ways to treat human disease.
Abbreviations: BMS, Bristol-Myers Squibb; Eat, arrested pharyngeal pumping; Egl-c, egg-laying constitutive; EMS, ethyl methanesulfonate; G-αq, G-protein alpha subunit; GAPs, GTPase activating proteins; (gf), gain-of-function; Gpa1, (the G-protein alpha subunit); GPCR, G-protein coupled receptor; GTP, guanosine triphosphate; (lf), loss-of-function; M3, muscarinic receptor type 3; PKC, protein kinase C; RGS, regulators of G-protein signaling; SNP, single nucleotide polymorphism; UI, urinary incontinence; Unc, uncoordinated motion
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