Fleming J V, Wang T C
Department of Medicine, Harvard Medical School, and Gastrointestinal Unit, Massachusetts General Hospital, Boston, MA 02114, USA.
Mol Cell Biol. 2000 Jul;20(13):4932-47. doi: 10.1128/MCB.20.13.4932-4947.2000.
Control of enzymatic function by peptide hormones can occur at a number of different levels and can involve diverse pathways that regulate cleavage, intracellular trafficking, and protein degradation. Gastrin is a peptide hormone that binds to the cholecystokinin B-gastrin receptor and regulates the activity of L-histidine decarboxylase (HDC), the enzyme that produces histamine. Here we show that gastrin can increase the steady-state levels of at least six HDC isoforms without affecting HDC mRNA levels. Pulse-chase experiments indicated that HDC isoforms are rapidly degraded and that gastrin-dependent increases are due to enhanced isoform stability. Deletion analysis identified two PEST domains (PEST1 and PEST2) and an intracellular targeting domain (ER2) which regulate HDC protein expression levels. Experiments with PEST domain fusion proteins demonstrated that PEST1 and PEST2 are strong and portable degradation-promoting elements which are positively regulated by both gastrin stimulation and proteasome inhibition. A chimeric protein containing the PEST domain of ornithine decarboxylase was similarly affected, indicating that gastrin can regulate the stability of other PEST domain-containing proteins and does so independently of antizyme/antizyme inhibitor regulation. At the same time, endoplasmic reticulum localization of a fluorescent chimera containing the ER2 domain of HDC was unaltered by gastrin stimulation. We conclude that gastrin stabilization of HDC isoforms is dependent upon two transferable and sequentially unrelated PEST domains that regulate degradation. These experiments revealed a novel regulatory mechanism by which a peptide hormone such as gastrin can disrupt the degradation function of multiple PEST-domain-containing proteins.
肽类激素对酶功能的调控可发生在多个不同层面，且可能涉及多种调节切割、细胞内运输及蛋白质降解的途径。胃泌素是一种肽类激素，它与胆囊收缩素B - 胃泌素受体结合，并调节L - 组氨酸脱羧酶（HDC）的活性，HDC是产生组胺的酶。在此我们表明，胃泌素可增加至少六种HDC同工型的稳态水平，而不影响HDC mRNA水平。脉冲追踪实验表明，HDC同工型迅速降解，且胃泌素依赖性增加是由于同工型稳定性增强所致。缺失分析鉴定出两个PEST结构域（PEST1和PEST2）以及一个调节HDC蛋白表达水平的细胞内靶向结构域（ER2）。对PEST结构域融合蛋白的实验表明，PEST1和PEST2是强大且可转移的促进降解元件，它们受到胃泌素刺激和蛋白酶体抑制的正向调节。含有鸟氨酸脱羧酶PEST结构域的嵌合蛋白也受到类似影响，这表明胃泌素可调节其他含PEST结构域蛋白的稳定性，且这种调节独立于抗酶/抗酶抑制剂调节。同时，胃泌素刺激并未改变含有HDC的ER2结构域的荧光嵌合体在内质网的定位。我们得出结论，胃泌素对HDC同工型的稳定作用依赖于两个调节降解的可转移且序列无关的PEST结构域。这些实验揭示了一种新的调节机制，通过该机制，诸如胃泌素之类的肽类激素可破坏多种含PEST结构域蛋白的降解功能。
