Webb Christina K, McCudden Christopher R, Willard Francis S, Kimple Randall J, Siderovski David P, Oxford Gerry S
Department of Cell and Molecular Physiology and UNC Neuroscience Center, University of North Carolina, Chapel Hill, North Carolina, USA.
J Neurochem. 2005 Mar;92(6):1408-18. doi: 10.1111/j.1471-4159.2004.02997.x.
The GoLoco motif is a short polypeptide sequence found in G-protein signaling regulators such as regulator of G-protein signaling proteins type 12 and 14 and activator of G-protein signaling protein type 3. A unique property of the GoLoco motifs from these three proteins is their preferential interaction with guanosine diphosphate (GDP)-bound Galpha(i1), Galpha(i3) and, sometimes, Galpha(i2) subunits over Galpha(o) subunits. This interaction prevents both spontaneous guanine nucleotide release and reassociation of Galpha(i)-GDP with Gbetagamma. We utilized this property of the GoLoco motif to examine dopamine (D2 and D3) and somatostatin receptor coupling to G-protein-regulated inwardly rectifying potassium (GIRK) channels in mouse AtT20 cells. GoLoco motif peptides had no effect on either basal channel activity or the initial responses to agonists, suggesting that the GoLoco motif cannot disrupt pre-formed G-protein heterotrimers. GoLoco motif peptides did, however, interfere with human D2((short)) receptor coupling to GIRK channels as demonstrated by the progressively diminished responses after repeated agonist application. This behavior is consistent with some form of compartmentalization of D2 receptors and GIRK channels such that Gbetagamma subunits, freed by local receptor activation and prevented from reforming a heterotrimeric complex, are not functionally constrained within the receptor-channel complex and thus are unable to exert a persistent activating effect. In contrast, GoLoco motif peptides had no effect on either D3 or somatostatin coupling to GIRK channels. Our results suggest that GoLoco motif-based peptides will be useful tools in examining the specificity of G-protein-coupled receptor-effector coupling.
GoLoco基序是一种短多肽序列，存在于G蛋白信号调节因子中，如12型和14型G蛋白信号调节蛋白以及3型G蛋白信号激活蛋白。这三种蛋白质的GoLoco基序的一个独特特性是，它们优先与结合鸟苷二磷酸（GDP）的Gα(i1)、Gα(i3)亚基，有时还与Gα(i2)亚基相互作用，而不是与Gα(o)亚基相互作用。这种相互作用既阻止了鸟嘌呤核苷酸的自发释放，也阻止了Gα(i)-GDP与Gβγ的重新结合。我们利用GoLoco基序的这一特性，研究了小鼠AtT20细胞中多巴胺（D2和D3）和生长抑素受体与G蛋白调节内向整流钾（GIRK）通道的偶联。GoLoco基序肽对基础通道活性或对激动剂的初始反应均无影响，这表明GoLoco基序不能破坏预先形成的G蛋白异源三聚体。然而，如反复应用激动剂后反应逐渐减弱所表明的，GoLoco基序肽确实干扰了人类D2（短）受体与GIRK通道的偶联。这种行为与D2受体和GIRK通道的某种形式的区室化一致，即由局部受体激活释放并阻止重新形成异源三聚体复合物的Gβγ亚基，在受体-通道复合物内没有功能限制，因此无法发挥持续的激活作用。相比之下，GoLoco基序肽对D3或生长抑素与GIRK通道的偶联均无影响。我们的结果表明，基于GoLoco基序的肽将是研究G蛋白偶联受体-效应器偶联特异性的有用工具。
