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Published September 16, 2005 | Published
Journal Article Open

A Peptide Core Motif for Binding to Heterotrimeric G Protein α Subunits


Recently, in vitro selection using mRNA display was used to identify a novel peptide sequence that binds with high affinity to G{alpha}i1. The peptide was minimized to a 9-residue sequence (R6A-1) that retains high affinity and specificity for the GDP-bound state of G{alpha}i1 and acts as a guanine nucleotide dissociation inhibitor (GDI). Here we demonstrate that the R6A-1 peptide interacts with G{alpha} subunits representing all four G protein classes, acting as a core motif for G{alpha} interaction. This contrasts with the consensus G protein regulatory(GPR) sequence, a 28-mer peptide GDI derived from the GoLoco (G{alpha}i/0-Loco interaction)/GPR motif that shares no homology with R6A-1 and binds only to G{alpha}i1-3 in this assay. Binding of R6A-1 is generally specific to the GDP-bound state of the G{alpha} subunits and excludes association with G{beta}{gamma}. R6A-G{alpha}i1 complexes are resistant to trypsin digestion and exhibit distinct stability in the presence of Mg2+, suggesting that the R6A and GPR peptides exert their activities using different mechanisms. Studies using G{alpha}i1/G{alpha}s chimeras identify two regions of G{alpha}i1 (residues 1–35 and 57–88) as determinants for strong R6A-Gi{alpha}1 interaction. Residues flanking the R6A-1 peptide confer unique binding properties, indicating that the core motif could be used as a starting point for the development of peptides exhibiting novel activities and/or specificity for particular G protein subclasses or nucleotide-bound states.

Additional Information

© 2005 the American Society for Biochemistry and Molecular Biology. Received for publication, July 20, 2005. Originally published In Press as doi:10.1074/jbc.C500319200 on July 28, 2005. We thank Dr. David S. Waugh (NCI-Frederick, National Institutes of Health) for providing the original pDW363 in vivo biotinylation vector, Prof. Nikolai O. Artemyev and Dr. Michael Natochin (University of Iowa) for providing the G{alpha}i1/G{alpha}s chimeric constructs, Dr. Yuri K. Peterson (Duke University) for advice on characterizing the R6A-derived peptides, and Dr. Bradley M. Denker (Harvard Institutes of Medicine) and Prof. Carl Schmidt (University of Delaware) for their suggestions on G protein translation. We are also grateful to Dr. Terry T. Takahashi and Christine T. Ueda for comments on the paper. This work was supported in part by Grant I1229 from the Welch Foundation (to S.R.S.) and National Institutes of Health Grant RO1 GM 60416 and the Beckman Foundation (to R.W.R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. [W.W.J. was] [s]upported in part by a Department of Defense National Defense Science and Engineering Graduate. Fellowship.

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August 22, 2023
October 16, 2023