Garcia-Marcos, M. | Search bioRxiv

bioRxiv2020

Optogenetic activation of heterotrimeric Gi proteins by LOV2GIVe - a rationally engineered modular protein

Heterotrimeric G-proteins are signal transducers that mediate the action of many natural extracellular stimuli as well as of many therapeutic agents. Non-invasive approaches to manipulate the activity of G-proteins with high precision are crucial to understand their regulation in space and time. Here, we engineered LOV2GIVe, a modular protein that allows the activation of Gi proteins with blue light. This optogenetic construct relies on a versatile design that differs from tools previously developed for similar purposes, i.e. metazoan opsins, which are light-activated GPCRs. To make LOV2GIVe, we fused a peptide derived from a non-GPCR protein that activates Gi (but not Gs, Gq, or G12) to a small plant protein domain, such that light uncages the G-protein activating module. Targeting LOV2GIVe to cell membranes allowed for light-dependent activation of Gi proteins in different experimental systems. In summary, LOV2GIVe expands the armamentarium and versatility of tools available to manipulate heterotrimeric G-protein activity. GRAPHICAL SUMMARY O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/253781v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1870610org.highwire.dtl.DTLVardef@19f2b2dorg.highwire.dtl.DTLVardef@cb9e57org.highwire.dtl.DTLVardef@88924_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Receptor tyrosine kinases activate heterotrimeric G proteins via phosphorylation within the interdomain cleft of Gαi

The molecular mechanisms by which receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major signaling hubs in eukaryotes, independently relay signals across the plasma membrane have been extensively characterized. How these hubs crosstalk has been a long-standing question, but answers remain elusive. Using linear-ion-trap mass spectrometry in combination with biochemical, cellular, and computational approaches, we unravel a mechanism of activation of heterotrimeric G proteins by RTKs and chart the key steps that mediate such activation. Upon growth factor stimulation, the guanine-nucleotide exchange modulator, GIV, dissociates Gi*{beta}{gamma} trimers, scaffolds monomeric Gi with RTKs, and facilitates the phosphorylation on two tyrosines located within the inter-domain cleft of Gi. Phosphorylation triggers the activation of Gi and inhibits second messengers (cAMP). Tumor-associated mutants reveal how constitutive activation of this pathway impacts cells decision to go vs. grow. These insights define a tyrosine-based G protein signaling paradigm and reveal its importance in eukaryotes. Significance StatementGrowth factors and heterotrimeric G proteins are two of the most widely studied signaling pathways in eukaryotes; their crosstalk shapes some of the most fundamental cellular responses in both health and disease. Although mechanisms by which G protein pathways transactivate growth factor RTKs has been well-defined, how the reverse may happen is less understood. This study defines the key steps and cellular consequences of a fundamental mechanism of signal crosstalk that enables RTKs to transactivate heterotrimeric G protein, Gi. Mutations found in tumors shed light on how derailing this mechanism impacts tumor cell behavior. Thus, findings not only show how cells integrate extracellular signals via pathway crosstalk, but also demonstrate the relevance of this pathway in cancers.

biochemistry↗