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J. Biol. Chem., Vol. 280, Issue 37, 32057-32060, September 16, 2005

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A Peptide Core Motif for Binding to Heterotrimeric G Protein Subunits^*

William W. Ja1, Anirban Adhikari, Ryan J. Austin, Stephen R. Sprang¶, and Richard W. Roberts2

From the Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125 and the Department of Biochemistry and Molecular Biophysics Graduate Program and ^¶The Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Recently, in vitro selection using mRNA display was used to identify a novel peptide sequence that binds with high affinity to G_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_i1 and acts as a guanine nucleotide dissociation inhibitor (GDI). Here we demonstrate that the R6A-1 peptide interacts with G subunits representing all four G protein classes, acting as a core motif for G interaction. This contrasts with the consensus G protein regulatory(GPR) sequence, a 28-mer peptide GDI derived from the GoLoco (G_i/0-Loco interaction)/GPR motif that shares no homology with R6A-1 and binds only to G_i1-3 in this assay. Binding of R6A-1 is generally specific to the GDP-bound state of the G subunits and excludes association with G. R6A-G_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_i1/G_s chimeras identify two regions of G_i1 (residues 1–35 and 57–88) as determinants for strong R6A-G_i1 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.

Received for publication, July 20, 2005

^* 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.

¹ Supported in part by a Department of Defense National Defense Science and Engineering Graduate Fellowship.

² To whom correspondence should be addressed. Tel.: 626-395-2321; Fax: 626-568-9430; E-mail: rroberts{at}caltech.edu.

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