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Papers In Press, published online ahead of print October 6, 2003
Department of Biochemistry, The Howard Hughes Medical Institute, Dallas, TX 75390-9050
Corresponding Author: stephen.sprang{at}utsouthwestern.edu
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(AGS3-C). The entire C-terminal domain as well as certain peptides corresponding to individual GPR motifs of AGS3 bind to G
J. Biol. Chem, 10.1074/jbc.M306300200
Submitted on June 15, 2003
Revised on September 9, 2003
Accepted on October 6, 2003
Thermodynamic characterization of the binding of AGS3 and peptides derived from AGS3 with G
i1
i1 and inhibit the binding of GTP by stabilizing the GDP-bound conformation of Gi1. The stoichiometry, free energy, enthalpy and dissociation constant for binding of AGS3-C to Gi1 were determined using isothermal titration calorimetry (ITC). AGS3-C possesses two apparent high-affinity (Kd ~ 20 nM) and two apparent low-affinity (Kd ~ 300 nM) binding sites for Gi1. Upon deletion of the C-terminal GPR motif from AGS3-C, the remaining sites are approximately equivalent with respect to their affinity (Kd ~ 400 nM) for Gi1. 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) bind to Gai1 with Kd values in the range of 1-8 
M. Although GPR1, GPR2 and GPR4 inhibit the binding of the fluorescent GTP analog BODIPY-FL-GTP
S to Gi1, GPR3 does not. However, addition of N- and C- terminal flanking residues to the GPR3 GoLoco core increases its affinity for Gi1 and confers GDI activity similar to that of AGS3-C itself. Similar increases are 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 towards Gi1 even though the amino acid sequences of these residues are not conserved among the GPR repeats.
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