Regulation Of GTP-binding Proteins

ADP-核糖基化因子（ARF）是调节内质网和高尔基体（和其他地方）中囊泡运输的GTP结合蛋白。ARF功能需要GTP结合的活性和GDP结合的非活性形式之间的调节性交替。GTP结合由鸟嘌呤核苷酸交换蛋白（GEP）催化，其中一些被BFA（一种抑制蛋白分泌并导致高尔基体池可逆崩解的药物）抑制，其中一些是BFA抗性的。在后者是家庭的~50-kDa的cytohesins，第四个成员，其中克隆和特点的小组。 两个BFA抑制的GEPs（BIG 1和BIG 2）已被纯化作为~670-kDa大分子复合物的组分。随后进行克隆，然后用重组蛋白进行结构-功能研究。来自培养细胞的内源性BIG 1和BIG 2与特异性抗体的免疫沉淀物也沉淀70-75%的其他蛋白质。这两个非常相似的BIGs在酵母双杂交实验中表现得非常不同，其中BIG 1与FKBP相互作用，BIG 2与RIa相互作用，RIa是环AMP活化蛋白激酶A（PKA）的调节亚基。FKBP 13是FK 506结合亲免蛋白家族的成员，与更好地已知的FKBP 12不同，FKBP 13不结合或抑制钙调磷酸酶。与广泛使用的免疫抑制药物FK 506一起孵育Jurkat细胞，增加了BIG 1，BIG 2和ARF与高尔基体和其他膜的结合，就像结构相关的激动剂一样。结构相关的拮抗剂、环孢菌素A或雷帕霉素（不通过FKBP起作用的免疫抑制剂）均未观察到作用。这些发现与FKBP 13和FK 506在囊泡运输中的作用一致，通过BIG 1影响ARF活性。 BIG 2与RIa的相互作用通过体外翻译的BIG 2和RIa以及来自培养的Hep G2细胞的内源性蛋白质的免疫共沉淀来证实。用提高细胞环AMP浓度的试剂孵育细胞导致BIG 2（和BIG 1）易位到高尔基体和其他膜。使用28个BIG 2的缺失突变体，鉴定了与PKA的四个R亚基中的一个或多个相互作用的分子的三个区域。序列的螺旋轮投影揭示了AKAP（A激酶锚定蛋白）的潜在两亲性螺旋结构特征。由环AMP控制的广谱信号传导事件需要PKA及其底物沿着与参与特定细胞内功能的其他分子的区室化。这些发现与BIG 2在协调cAMP和ARF调节途径中的作用一致。

ADP-ribosylation factors (ARFs) are GTP-binding 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), some of which are inhibited by BFA (a drug that inhibits protein sectretion and causes reversible disintegration of Golgi cisternae) and some of which are BFA-resistant. Among the latter is the family of ~50-kDa cytohesins, the fourth member, of which was cloned and characterized by the group. Two BFA-inhibited GEPs (BIG1 and BIG2) had been purified by the group as components of ~670-kDa macromolecular complex. Subsequent cloning was followed by structure-function studies with recombinant proteins. Immunoprecipitates of endogeneous BIG1 and BIG2 from cultured cells with specific antibodies precipitated also 70-75% of the other protein. The two very similar BIGs behaved quite differently in yeast two-hybrid experiments, where BIG1 interacted with an FKBP, and BIG2 with RIa, a regulatory subunit of cyclic AMP-activated protein kinase A (PKA). FKBP13 is a member of the FK506-binding family of immunophilins that, unlike the better known FKBP12, does not bind or inhibit calcineurin. Incubation of Jurkat cells with FK506, a widely used immunosuppressive drug, increased binding of BIG1, BIG2, and ARF to Golgi and other membranes as did a structurally related agonist. No effect was seen with a structurally related antagonist,or with cyclosporin A, or rapamycin, immunosuppressants that do not act via an FKBP. These findings are consistent with the role for FKBP13 and FK506 in vesicular trafficking, influencing ARF activity through BIG1. BIG2 interaction with RIa was confirmed by co-immunoprecipitation of in vitro-translated BIG2 and Ria, as well as of the endogenous proteins from cultured Hep G2 cells. Incubation of cells with agents that raised cell cyclic AMP concentration resulted in translocation of BIG2 (and BIG1) to Golgi and other membranes. Using 28 deletion mutants of BIG2, three regions of the molecule that interacted with one or more of the four R subunits of PKA were identified. Helical wheel projections of the sequences revealed potential amphipathic helical structures characteristic of AKAPs (A kinase-anchoring proteins). The broad spectrum of signalling events controlled by cyclic AMP requires compartmentalization of PKA and its substrate(s) along with the other molecules that are involved in specific intracellular functions. These findings are consistent with a role for BIG2 in coordinating cAMP and ARF regulatory pathways.