Turning G proteins on and off: peptide modulators of signaling

Heterotrimeric G proteins play a critical role in communicating extracellular signals to intracellular signal transduction pathways through transmembrane receptors. Previously, the 9‐residue R6A‐1 peptide was identified as a core motif for binding to heterotrimeric G protein α subunits. R6A‐1 acts as a guanine nucleotide dissociation inhibitor for Gα i1 and blocks association with G βγ . In vitro selection with a new mRNA display peptide library—based on the R6A‐1 core motif sequence—identified thousands of unique peptide sequences that bind to Gα i1 . The effects of these peptides on G protein signaling in vivo was observed by electrophysiology, using cell lines expressing GIRK channels—potassium channels that are activated by released G βγ subunits. Peptides were identified that enhance or repress basal levels of signaling, demonstrating the ability to directly turn G proteins on and off. In vitro binding studies show that the peptides are able to disrupt or stabilize G protein heterotrimers, in accord with the observed in vivo activities. The R6A‐1 peptide, which had no effect on basal channel activity, weakened responses to repeated agonist application. Hence, we have identified ligands that act as direct activators, inhibitors, and attenuators of G protein signaling. These peptide modulators should be useful as tools for probing G protein function and as leads for developing novel molecules with new selectivities and/or activities. Funding from the Beckman Foundation and NIH GM60416 (R.W.R.), Howard Hughes Medical Institute and NIMH MH63981 (L.Y.J., in the Silvio Conte Center of Neuroscience at UCSF), European Molecular Biology Organization (O.W.), and a DOD National Defense Science and Engineering Graduate Fellowship (W.W.J.).