Gα selectivity and inhibitor function of the multiple GoLoco motif protein GPSM2/LGN
Gα selectivity and inhibitor function of the multiple GoLoco motif protein GPSM2/LGN
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DOI：
10.1016/j.bbamcr.2005.05.002
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发表时间：
2005-09-10
影响因子：
5.1
通讯作者：
Siderovski, DP
中科院分区：
文献类型：
--
作者：
McCudden, CR;Willard, FS;Siderovski, DP
GPSM 2（G蛋白信号调节剂2;也称为LGN或哺乳动物Pins）是一种调节有丝分裂纺锤体组织和细胞分裂的蛋白质。GPSM 2含有七个三肽重复序列（TPR）和四个G alpha（i/o）-Loco（GoLoco）基序。GPSM 2对G α（o）-和G α（i）-亚基都具有鸟嘌呤核苷酸解离抑制剂（GDI）活性;然而，尚未描述其单个GoLoco基序的系统分析。我们分析了来自GPSM 2的四个单独的GoLoco基序中的每一个，评估了它们对G α（i1）、G α（i2）、G α（i3）和G α（o）的相对结合亲和力和GDI效力。将四个GPSM 2 GoLoco基序（长度为36-43个氨基酸）中的每一个在细菌中表达为GST融合蛋白并纯化至同质。通过表面等离子体共振评估了四种GST-GoLoco基序中的每一种与G α（i1）-、G α（o）-和G α（s）-亚基的结合;所有基序都与G α（iI）结合，但对G α（o）表现出低亲和力。通过基于荧光的核苷酸结合测定评估GDI活性，揭示了所有四个GoLoco基序作为G α（i1）、G α（i2）和G α（i3）的GDI起作用。与我们的结合研究一致，GPSM 2 GoLoco基序对Ga-0的GDI活性显著低于对G α（i 1）的GDI活性，表明GPSM 2的体内靶点最可能是Ga i-亚基。(c)2005 Elsevier B. V.保留所有权利。
GPSM2 (G-protein signalling modulator 2; also known as LGN or mammalian Pins) is a protein that regulates mitotic spindle organization and cell division. GPSM2 contains seven tetratricopeptide repeats (TPR) and four G alpha(i/o)-Loco (GoLoco) motifs. GPSM2 has guanine nucleotide dissociation inhibitor (GDI) activity towards both G alpha(o)- and G alpha(i)-subunits; however, a systematic analysis of its individual GoLoco motifs has not been described. We analyzed each of the four individual GoLoco motifs from GPSM2, assessing their relative binding affinities and GDI potencies for G alpha(i1), G alpha(i2), and G alpha(i3) and G alpha(o). Each of the four GPSM2 GoLoco motifs (36-43 amino acids in length) was expressed in bacteria as a GST-fusion protein and purified to homogeneity. The binding of each of the four GST-GoLoco motifs to G alpha(i1)-, G alpha(o)-, and G alpha(s)-subunits was assessed by surface plasmon resonance; all of the motifs bound G alpha(iI), but exhibited low affinity towards G alpha(o). GDI activity was assessed by a fluorescence-based nucleotide-binding assay, revealing that all four GoLoco motifs are functional as GDIs for G alpha(i1), G alpha(i2), and G alpha(i3). Consistent with our binding studies, the GDI activity of GPSM2 GoLoco motifs on Ga-o was significantly lower than that toward G alpha(i1), suggesting that the in vivo targets of GPSM2 are most likely to be Gai-subunits. (c) 2005 Elsevier B.V. All rights reserved.