A biochemical and genetic discovery pipeline identifies PLC?4b as a nonreceptor activator of heterotrimeric G-proteins.
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ABSTRACT: Recent evidence has revealed that heterotrimeric G-proteins can be activated by cytoplasmic proteins that share an evolutionarily conserved sequence called the G?-binding-and-activating (GBA) motif. This mechanism provides an alternative to canonical activation by G-protein-coupled receptors (GPCRs) and plays important roles in cell function, and its dysregulation is linked to diseases such as cancer. Here, we describe a discovery pipeline that uses biochemical and genetic approaches to validate GBA candidates identified by sequence similarity. First, putative GBA motifs discovered in bioinformatics searches were synthesized on peptide arrays and probed in batch for G?i3 binding. Then, cDNAs encoding proteins with G?i3-binding sequences were expressed in a genetically-modified yeast strain that reports mammalian G-protein activity in the absence of GPCRs. The resulting GBA motif candidates were characterized by comparison of their biochemical, structural, and signaling properties with those of all previously described GBA motifs in mammals (GIV/Girdin, DAPLE, Calnuc, and NUCB2). We found that the phospholipase C?4 (PLC?4) GBA motif binds G-proteins with high affinity, has guanine nucleotide exchange factor activity in vitro, and activates G-protein signaling in cells, as indicated by bioluminescence resonance energy transfer (BRET)-based biosensors of G-protein activity. Interestingly, the PLC?4 isoform b (PLC?4b), which lacks the domains required for PLC activity, bound and activated G-proteins more efficiently than the full-length isoform a, suggesting that PLC?4b functions as a G-protein regulator rather than as a PLC. In summary, we have identified PLC?4 as a nonreceptor activator of G-proteins and established an experimental pipeline to discover and characterize GBA motif-containing proteins.
SUBMITTER: Maziarz M
PROVIDER: S-EPMC6222092 | biostudies-literature | 2018 Nov
REPOSITORIES: biostudies-literature
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