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J. Biol. Chem., Vol. 278, Issue 51, 51825-51832, December 19, 2003

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Thermodynamic Characterization of the Binding of Activator of G Protein Signaling 3 (AGS3) and Peptides Derived from AGS3 with G_i1^*

Anirban Adhikari and Stephen R. Sprang¶

From the Department of Biochemistry and Molecular Biophysics Graduate Program and The Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, Texas 75390

Activator of G protein signaling 3 (AGS3) is a guanine nucleotide dissociation inhibitor (GDI) that contains four G protein regulatory (GPR) or GoLoco motifs in its C-terminal domain. The entire C-terminal domain (AGS3-C) as well as certain peptides corresponding to individual GPR motifs of AGS3 bound to G_i1 and inhibited the binding of GTP by stabilizing the GDP-bound conformation of G_i1. The stoichiometry, free energy, enthalpy, and dissociation constant for binding of AGS3-C to G_i1 were determined using isothermal titration calorimetry. AGS3-C possesses two apparent high affinity (K_d 20 nM) and two apparent low affinity (K_d 300 nM) binding sites for G_i1. Upon deletion of the C-terminal GPR motif from AGS3-C, the remaining sites were approximately equivalent with respect to their affinity (K_d 400 nM) for G_i1. Peptides corresponding to each of the four GPR motifs of AGS3 (referred to as GPR1, GPR2, GPR3, and GPR4, respectively, going from N to C terminus) bound to G_i1 with K_d values in the range of 1-8 µM. Although GPR1, GPR2, and GPR4 inhibited the binding of the fluorescent GTP analog BODIPY-FL-guanosine 5'-3-O-(thio)triphosphate to G_i1, GPR3 did not. However, addition of N- and C-terminal flanking residues to the GPR3 GoLoco core increased its affinity for G_i1 and conferred GDI activity similar to that of AGS3-C itself. Similar increases were observed for extended GPR2 and extended GPR1 peptides. Thus, while the tertiary structure of AGS3 may affect the affinity and activity of the GPR motifs contained within its sequence, residues outside of the GPR motifs strongly potentiate their binding and GDI activity toward G_i1 even though the amino acid sequences of these residues are not conserved among the GPR repeats.

Received for publication, June 15, 2003 , and in revised form, September 9, 2003.

^* This work was supported by National Institutes of Health Grant DK46371, Robert A. Welch Foundation Grant I-1229, and the John W. and Rhonda K. Pate Professorship in Biochemistry (to S. R. S.). 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.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry, The Howard Hughes Medical Inst., The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390. Tel.: 214-648-5008; Fax: 214-648-6336; E-mail: Stephen.Sprang{at}UTSouthwestern.edu.

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