Ric-8B Is a GTP-dependent G Protein αs Guanine Nucleotide Exchange Factor
Ric-8B Is a GTP-dependent G Protein αs Guanine Nucleotide Exchange Factor
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DOI：
10.1074/jbc.m110.163675
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发表时间：
2011-06-03
影响因子：
4.8
通讯作者：
Tall, Gregory G.
中科院分区：
文献类型：
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作者：
Chan, PuiYee;Gabay, Meital;Tall, Gregory G.
ric-8（抗胆碱酯酶8抑制剂）基因在多种G蛋白信号通路中具有积极作用，包括G α (q)和G α (s)对神经传递的调节，（不对称）细胞分裂过程中G α (i)依赖的有丝分裂纺锤体定位，以及G α (olf)依赖的气味受体信号传导。哺乳动物的ric8活性分为两个基因，ric8 - 8a和ric8 - 8b。ric8a是G α (i)/ α (q)/ α(12/13)亚基的鸟嘌呤核苷酸交换因子（GEF）。Ric-8B可能作为G α (s)级GEF激活剂增强G(s)信号，但没有证据表明Ric-8B具有GEF活性。本文纯化了两个Ric-8B亚型，发现它们是G α亚基GDP释放因子/GEFs。在HeLa细胞中，全长Ric-8B （Ric-8BFL）结合内源性表达G α s和少量G α (q)和G α(13)。Ric-8BFL刺激鸟苷5′-3- o -（硫）三磷酸（GTP γ S）与这些亚基和G α (olf)结合，而Ric-8B δ 9异构体仅刺激G α (S)（短）GTP γ S结合。Michaelis-Menten实验表明，ric8bfl提高了G α (s)稳态水解的V-max， GTP与G α (s)结合的表观K-m值从约385 nM提高到约42 μ m。ric8bfl催化的G α s交换反应的方向依赖于GTP。在低于k -m GTP时，尽管Ric-BFL是快速的GDP释放加速器，但对交换具有抑制作用。Ric-8BFL与无核苷酸的G α s紧密结合，需要接近k -m的GTP水平才能解离Ric-8B。无G α核苷酸中间体，释放游离的Ric-8B和G α - gtp。ric - 8bfl催化的核苷酸交换可能正向进行，在细胞中产生G α - gtp。
ric-8 (resistance to inhibitors of cholinesterase 8) genes have positive roles in variegated G protein signaling pathways, including G alpha(q) and G alpha(s) regulation of neurotransmission, G alpha(i)-dependent mitotic spindle positioning during (asymmetric) cell division, and G alpha(olf)-dependent odorant receptor signaling. Mammalian Ric-8 activities are partitioned between two genes, ric-8A and ric-8B. Ric-8A is a guanine nucleotide exchange factor (GEF) for G alpha(i)/alpha(q)/alpha(12/13) subunits. Ric-8B potentiated G(s) signaling presumably as a G alpha(s)-class GEF activator, but no demonstration has shown Ric-8B GEF activity. Here, two Ric-8B isoforms were purified and found to be G alpha subunit GDP release factor/GEFs. In HeLa cells, full-length Ric-8B (Ric-8BFL) bound endogenously expressed G alpha s and lesser amounts of G alpha(q) and G alpha(13). Ric-8BFL stimulated guanosine 5 '-3-O-(thio) triphosphate (GTP gamma S) binding to these subunits andG alpha(olf), whereas the Ric-8B Delta 9 isoform stimulated G alpha(s) (short) GTP gamma S binding only. Michaelis-Menten experiments showed that Ric-8BFL elevated the V-max of G alpha(s) steady stateGTPhydrolysis and the apparent K-m values of GTP binding to G alpha(s) from similar to 385 nM to an estimated value of similar to 42 mu M. Directionality of the Ric-8BFL-catalyzed G alpha s exchange reaction was GTP-dependent. At sub-K-m GTP, Ric-BFL was inhibitory to exchange despite being a rapid GDP release accelerator. Ric-8BFL binds nucleotide-free G alpha s tightly, and near-K-m GTP levels were required to dissociate the Ric-8B.G alpha nucleotide-free intermediate to release free Ric-8B and G alpha-GTP. Ric-8BFL-catalyzed nucleotide exchange probably proceeds in the forward direction to produce G alpha-GTP in cells.