GTP-BINDING PROTEIN STRUCTURE/FUNCTION STUDIES

ADP-核糖化因子(ARF)是一种多功能、多结构域的蛋白质，它调节内质网和高尔基体(以及其他部位)中的囊泡运输。ARF功能需要GTP结合的活性形式和GDP结合的非活性形式之间的调节交替。GTP结合是由鸟嘌呤核苷酸交换蛋白(GEP)催化的。除了被BFA(一种抑制蛋白质分泌并导致高尔基脑池可逆性解体的药物)抑制的~200 kDa GEPs家族外，还有一个耐BFA的~55 kDa GEPs家族。它们都有所谓的Sec7结构域，约200个氨基酸，负责GEP的活性。细胞粘附素-1是通过与整合素的相互作用而克隆的，被该小组鉴定为BFA不敏感的ARF GEP。细胞粘附素-2的预测氨基酸序列(83%相同)仅在存在或不存在单一甘氨酸的情况下不同。该小组证明了存在两种不同的信使核糖核酸亚型，对于已知的三种细胞粘附素中的每一种都是以相同的方式不同，并鉴定出一种新的只有一种信使核糖核酸的细胞粘附素-4。为了阐明控制甘氨酸单链插入的机制(S)，比较了细胞粘附素-1和细胞粘附素-4的基因，发现除了细胞粘附素-1的一个额外的3个碱基的外显子外，两个基因在结构上有显著的相似性。额外的甘氨酸预计会对功能产生重大影响，但可能不会对GEP活性产生重大影响，因为去除PH结构域会取消PIP2对细胞粘素的激活，但不会对GEP活性产生影响。尽管细胞粘附素-4在GEP活性和基因结构上与其他的相似，但它在特定的淋巴细胞亚群中的分布不同，提示其功能不同。

ADP-ribosylation factors (ARFs) are multi-functional, multi-domain, proteins that regulate vesicular trafficking in the ER and Golgi (and elsewhere). ARF function requires the regulated alternation between GTP-bound active and GDP-bound inactive forms. GTP binding is catalyzed by guanine nucleotide-exchange proteins (GEPs). In addition to a family of ~200-kDa GEPs that are inhibited by BFA (a drug that inhibits protein secretion and causes reversible disintegration of Golgi cisternae) is a family of ~55-kDa GEPs that are BFA-resistant. All have so-called Sec7 domains of ~200 amino acids that are responsible for GEP activity. Cytohesin-1, cloned by its interaction with integrin, had been characterized by the group as a BFA-insensitive ARF GEP. Predicted amino acid sequences for cytohesin-2 (83% identical) differed only in the presence or absence of a single glycine. The group demonstrated the existence of two mRNA isoforms that differ in the same way for each of three know cytohesins and identified a new cytohesin-4 with only one mRNA. To elucidate mechanism(s) that control the single glycine insertion, genes for cytohesin-1 and -4 were compared, revealing remarkable similarity of structure except for an extra 3-bp exon in cytohesin-1. The extra glycine is expected to have major effects on function, but probably not on GEP activity, since removal of the PH domain abolishes cytohesin activation by PIP2 but not GEP activity. Although, cytohesin-4 resembles the others in its GEP activity and gene structure, it differed in distribution in specific lymphocyte subpopulations, suggestive of different functions.