Adhikari Anirban, Sprang Stephen R
Department of Biochemistry and Molecular Biophysics Graduate Program, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
J Biol Chem. 2003 Dec 19;278(51):51825-32. doi: 10.1074/jbc.M306300200. Epub 2003 Oct 6.
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 alpha i1 and inhibited the binding of GTP by stabilizing the GDP-bound conformation of G alpha i1. The stoichiometry, free energy, enthalpy, and dissociation constant for binding of AGS3-C to G alpha i1 were determined using isothermal titration calorimetry. AGS3-C possesses two apparent high affinity (Kd approximately 20 nm) and two apparent low affinity (Kd approximately 300 nm) binding sites for G alpha i1. Upon deletion of the C-terminal GPR motif from AGS3-C, the remaining sites were approximately equivalent with respect to their affinity (Kd approximately 400 nm) for G alpha 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 alpha i1 with Kd values in the range of 1-8 microm. Although GPR1, GPR2, and GPR4 inhibited the binding of the fluorescent GTP analog BODIPY-FL-guanosine 5'-3-O-(thio)triphosphate to G alpha i1, GPR3 did not. However, addition of N- and C-terminal flanking residues to the GPR3 GoLoco core increased its affinity for G alpha 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 alpha i1 even though the amino acid sequences of these residues are not conserved among the GPR repeats.
G蛋白信号转导激活因子3（AGS3）是一种鸟嘌呤核苷酸解离抑制剂（GDI），其C末端结构域包含四个G蛋白调节（GPR）或GoLoco基序。整个C末端结构域（AGS3-C）以及与AGS3的各个GPR基序相对应的某些肽与Gαi1结合，并通过稳定Gαi1的GDP结合构象来抑制GTP的结合。使用等温滴定量热法测定了AGS3-C与Gαi1结合的化学计量、自由能、焓和解离常数。AGS3-C对Gαi1具有两个明显的高亲和力（Kd约为20 nM）结合位点和两个明显的低亲和力（Kd约为300 nM）结合位点。从AGS3-C中删除C末端GPR基序后，其余位点对Gαi1的亲和力（Kd约为400 nM）大致相同。与AGS3的四个GPR基序（分别从N端到C端称为GPR1、GPR2、GPR3和GPR4）相对应的肽与Gαi1结合，Kd值在1-8 μM范围内。虽然GPR1、GPR2和GPR4抑制了荧光GTP类似物BODIPY-FL-鸟苷5'-3-O-（硫代）三磷酸与Gαi1的结合，但GPR3没有。然而，在GPR3 GoLoco核心的N端和C端添加侧翼残基增加了其对Gαi1的亲和力，并赋予了与AGS3-C本身类似的GDI活性。对于延伸的GPR2和延伸的GPR1肽也观察到了类似的增加。因此，虽然AGS3的三级结构可能会影响其序列中包含的GPR基序的亲和力和活性，但GPR基序之外的残基强烈增强了它们对Gαi1的结合和GDI活性，即使这些残基的氨基酸序列在GPR重复序列中并不保守。
