Regulation of Guanine Nucelotide Exchange in Inhibitory G Protein Alpha Subunit by Activator of G Protein Signaling 3 and Novel Regulatory Peptides




Adhikari, Anirban

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The release of GDP from the nucleotide binding pocket of G protein a subunit (Ga) is accelerated by guanine nucleotide exchange factors (GEFs) and inhibited by guanine nucleotide dissociation inhibitors (GDIs). The beta gamma subunit of heterotrimeric G protein and GoLoco motif are GDIs. Activator of G protein signaling 3 (AGS3), a GDI containing four GoLoco motifs in its C-terminal domain (AGS3-C), binds specifically to GDP-bound Gai1 and inhibits the binding of GTP to Gai1. The stoichiometry and dissociation constant for the binding of AGS3-C to Gai1, determined using isothermal titration calorimetry (ITC), indicates the presence of two apparent high affinity (Kd ~ 20 nM) and two apparent low affinity (Kd ~300 nM) binding sites for Gai1. Upon deletion of the C-terminal GoLoco motif from AGS3-C, the remaining sites are approximately equivalent with respect to their affinity (Kd ~ 400 nM) for Gai1. Peptides corresponding to each of the four GoLoco motifs of AGS3 (referred to as GPR1-4, going from the N to C terminus) bind to Gai1 with Kd values in the range of 1-8 muM. GPR1, GPR2, and GPR4 were active as GDIs, but GPR3 was inactive. However, addition of N and C terminal flanking residues to the GPR3 GoLoco core increases its affinity for Gai1 and conferred GDI activity to the level of AGS3 itself. R6A is a Gai1 GDP selective peptide that has GDI activity towards Gai1 despite no sequence similarity with the GoLoco motif. The 1.8 ?ystal structure of Gai1 GDP:R6A complex shows that switch II region of Gai1, which is disordered in the GDP bound state, adopts an ordered structure through its direct interactions with R6A. Binding of R6A orients residues 202 to 204 of switch II (GG-loop) towards the nucleotide binding pocket and within 3 ? switch I, which suggests that R6A mediates its activity by occluding the potential exit route of GDP. The structure of Gai1 GDP:R6A, when compared to the structures of GoLoco and G beta gamma bound Gai1 GDP, reveals similarity in the conformation of GG-loop among the GDI bound structures of Gai1. A mutation in R6A, designed to position the GG-loop away from the nucleotide binding pocket in Gai1 GDP:R6A complex, abolishes the GDI activity and instead confers GEF like activity to R6A. Thus, the conformation of the GG-loop is likely to be a crucial structural determinant of the rate of GDP release from Gai1.

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