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Specific inhibition of GPCR-independent G protein signaling by a rationally engineered protein

  1. Mikel Garcia-Marcosa,2
  1. aDepartment of Biochemistry, Boston University School of Medicine, Boston, MA 02118;
  2. bDepartment of Chemistry and Molecular Pharmacology, IRB Barcelona, 08028 Barcelona, Spain;
  3. cDepartment of Medicine, Boston University School of Medicine, Boston, MA 02118
  1. Edited by Stephen R. Sprang, University of Montana, Missoula, MT, and accepted by Editorial Board Member David E. Clapham October 18, 2017 (received for review May 14, 2017)

Significance

Dysregulation of signaling via heterotrimeric G proteins leads to pathogenesis. Thus, developing an efficient armamentarium to study G protein regulation is crucial for understanding the molecular basis of disease. The classical view of G protein activation as an exclusive function of G protein-coupled receptors has been challenged by the discovery of nonreceptor G protein activators. Dysregulation of a family of such nonreceptor activators has been linked to human disorders like cancer or birth defects, but the underlying mechanisms remain poorly understood due to the lack of experimental tools. Here, we use protein engineering to rationally design a genetically encoded inhibitor of these G protein activators and demonstrate its usefulness to block aberrant signaling in cancer cells and abrogate developmental malformations in animal embryos.

Abstract

Activation of heterotrimeric G proteins by cytoplasmic nonreceptor proteins is an alternative to the classical mechanism via G protein-coupled receptors (GPCRs). A subset of nonreceptor G protein activators is characterized by a conserved sequence named the Gα-binding and activating (GBA) motif, which confers guanine nucleotide exchange factor (GEF) activity in vitro and promotes G protein-dependent signaling in cells. GBA proteins have important roles in physiology and disease but remain greatly understudied. This is due, in part, to the lack of efficient tools that specifically disrupt GBA motif function in the context of the large multifunctional proteins in which they are embedded. This hindrance to the study of alternative mechanisms of G protein activation contrasts with the wealth of convenient chemical and genetic tools to manipulate GPCR-dependent activation. Here, we describe the rational design and implementation of a genetically encoded protein that specifically inhibits GBA motifs: GBA inhibitor (GBAi). GBAi was engineered by introducing modifications in Gαi that preclude coupling to every known major binding partner [GPCRs, Gβγ, effectors, guanine nucleotide dissociation inhibitors (GDIs), GTPase-activating proteins (GAPs), or the chaperone/GEF Ric-8A], while favoring high-affinity binding to all known GBA motifs. We demonstrate that GBAi does not interfere with canonical GPCR-G protein signaling but blocks GBA-dependent signaling in cancer cells. Furthermore, by implementing GBAi in vivo, we show that GBA-dependent signaling modulates phenotypes during Xenopus laevis embryonic development. In summary, GBAi is a selective, efficient, and convenient tool to dissect the biological processes controlled by a GPCR-independent mechanism of G protein activation mediated by cytoplasmic factors.

Footnotes

  • ?1A.L. and A.M. contributed equally to this work.

  • ?2To whom correspondence should be addressed. Email: mgm1{at}bu.edu.
  • Author contributions: A.L., A.M., M.M., V.D., I.D., and M.G.-M. designed research; A.L., A.M., M.M., V.D., M.P.P., P.P.P., J.B.B.-C., I.A.W., G.R.-D., and M.G.-M. performed research; J.B.B.-C. contributed new reagents/analytic tools; A.L., A.M., M.M., V.D., I.D., and M.G.-M. analyzed data; M.G.-M. wrote the paper; and M.G.-M. conceived the project.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission. S.R.S. is a guest editor invited by the Editorial Board.

  • This article contains supporting information online at www.danielhellerman.com/lookup/suppl/doi:10.1073/pnas.1707992114/-/DCSupplemental.

Published under the PNAS license.

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