REGULATION OF GTP-BINDING PROTEINS

ARF功能需要在gtp结合的活性形式和gdp结合的非活性形式之间进行调节。GTP的结合由鸟嘌呤核苷酸交换蛋白（GEPs）催化，GTP酶激活蛋白（gap）灭活GTP。这些研究中涉及的两种一般类型的GEPs是被BFA抑制的~200 kda蛋白（一种可逆干扰蛋白质分泌并导致高尔基池解体的药物）和较小的~50 kda的GEPs，它们具有BFA抗性。所有GEP都有大约200个氨基酸组成的所谓Sec7结构域，这些氨基酸负责GEP的活性及其BFA敏感性。该小组早些时候从牛脑中纯化了两个bfa抑制的GEPs (BIG1和BIG2)，它们在凝胶过滤时表现为~670 kDa的分子。特异的、亲和纯化的抗肽抗体的免疫沉淀表明，它们在很大程度上是相同的大分子复合物的组成部分。通过与整合素的相互作用克隆的50 kda的cytohesin-1，已经被研究小组证明是一个对bfa不敏感的ARF GEP。由独立cDNA克隆预测的细胞分裂素-2的氨基酸序列仅在存在或不存在单个甘氨酸时不同。该小组证明了存在两种mRNA同种异构体，它们以相同的方式不同于三种已知的细胞分裂素，并鉴定了一种只有一种mRNA的新的细胞分裂素-4。为了阐明控制单甘氨酸插入的机制，我们比较了细胞分裂素-1和-4的基因，发现除了细胞分裂素-1多一个3 bp外显子外，它们的结构非常相似。额外的甘氨酸改变了PH结构域的结构，导致细胞分裂素与特定磷酸肌苷相互作用的显著差异。目前正在研究调节细胞分裂素1、2和3的选择性剪接的机制，以及单个细胞分裂素的细胞特异性表达。在其他研究中，一个新的ARF GEP，不同于任何已知的，被克隆和表征。~100 kda蛋白包含在所有ARF GEPs中发现的Sec7结构域，但没有共享其他已识别的结构域结构。它的活性不受brefeldin A或磷脂的影响，它似乎优先作为ARF6的GEP发挥作用。这种mRNA在外周血白细胞、脑和脾脏中尤其丰富。免疫反应性内源性arf6和新的GEP100在囊泡结构中部分共定位，GEP的免疫荧光也与早期内体标志物EEA-1的免疫荧光一致。adp核糖基化因子结构域蛋白1 （ARD1）最初是在实验室中克隆的，与其他ARF不同的是，它存在一个46 kda的氨基末端延伸物（p5），该延伸物在其ARF结构域（p3）中充当gtpase激活蛋白（GAP）。与ARF的GAP类似，ARD1的GAP结构域含有锌指基序和精氨酸残基，这些残基对活性至关重要。它与其他ARF GAP的不同之处在于它与gtp结合域的共价结合以及它的GAP活性对ARD1的ARF结构域的特异性。据推测，arf在细胞内运输囊泡的形成及其从一个隔室到另一个隔室的运动中起关键作用。过表达和内源性ARD1都与高尔基体和溶酶体膜有关，与溶酶体的形成或功能以及高尔基体和溶酶体之间的蛋白质运输的作用一致。在酵母双杂交筛选中发现了ARD1与细胞分裂素-1的相互作用。细胞素-2在这个系统中不能相互作用，对ARD1的GEP活性远低于细胞素-1，尽管它们对ARF1的活性相同。在体外也显示出优先的物理相互作用，并且鉴定了负责细胞分裂素-1/ARD1相互作用特异性的残基。今年终于获得了“敲除”ARD1的小鼠，有关该蛋白生理功能的新线索正在出现。

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) and inactivation by GTPase-activating proteins (GAPs). Two general types of GEPs that have been involved in most of these studies are ~200-kDa proteins that are inhibited by BFA (a drug that reversibly interferes with protein secretion and causes disintegration of Golgi cisternae) and smaller ~50-kDa GEPs that are BFA-resistant. All GEPs have so-called Sec7 domains of ~200 amino acids that are responsible for the GEP activity, as well as its BFA sensitivity. The group had earlier purified, from bovine brain, two BFA-inhibited GEPs (BIG1 and BIG2) that behaved on gel filtration as molecules of ~670 kDa. Immunoprecipitation with specific, affinity-purified anti-peptide antibodies demonstrated that they are to a large extent components of the same macromolecular complexes. The 50-kDa cytohesin-1, cloned by its interaction with integrin, had been shown by the group to be a BFA-insensitive ARF GEP. Amino acid sequences for cytohesin-2 predicted by independent cDNA clones differ 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 known 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 alters PH domain structure resulting in significant differences in cytohesin interaction with specific phosphoinositides. Mechanisms that regulate the alternative splicing of cytohesins 1, 2, and 3, as well as the cell-specific expression of individual cytohesins are being investigated. In other studies, a novel ARF GEP, unlike any already known, was cloned and characterized. The ~100-kDa protein contains the Sec7 domain found in all ARF GEPs, but shares no other recognized domain structures. Its activity is not affected by brefeldin A or phospholipids, and it appears to function preferentiallly as a GEP for ARF6. The mRNA is particularly abundant in peripheral blood leukocytes, brain, and spleen. Immunoreactive endogenous ARF 6 and the new GEP100 were partially co-localized in vesicular structures and the GEP immunofluorescence also coincided with that of EEA-1, a marker for early endosomes. ADP-ribosylation factor domain protein 1 (ARD1), initially cloned in the laboratory, differs from other ARFs by the presence of a 46-kDa amino-terminal extension (p5), which acts as a GTPase-activating protein (GAP) for its ARF domain (p3). Similar to ARF GAPs, the GAP domain of ARD1 contains a zinc finger motif and arginine residues that are critical for activity. It differs from other ARF GAPs in its covalent association with the GTP-binding domain and the specificity of its GAP activity for the ARF domain of ARD1. ARFs are presumed to play a key role in the formation of intracellular transport vesicles and in their movement from one compartment to another. Both overexpressed and endogenous ARD1 were associated with Golgi and lysosomal membranes, consistent with a role in the formation or function of lysosomes and in protein trafficking bettween Golgi and lysosomes. Interaction of ARD1 and cytohesin-1 was found in a yeast two-hybrid screen. Cytohesin-2 failed to interact in this system and had much less GEP activity toward ARD1 than did cytohesin-1, although they were equally active with ARF1. Preferential physical interaction was also shown in vitro and residues responsible for specificity of the cytohesin-1/ARD1 interaction were identified. ARD1 "knock-out" mice were finally obtained this year and new clues to the physiological function of this protein are emerging.