Stoller T J, Shields D
Department of Anatomy, Albert Einstein College of Medicine, Bronx, New York 10461.
J Cell Biol. 1988 Dec;107(6 Pt 1):2087-95. doi: 10.1083/jcb.107.6.2087.
Somatostatin (SRIF) is a 14-amino acid peptide hormone that is synthesized as part of a larger precursor, preproSRIF, consisting of a signal peptide and a proregion of 80-90 amino acids. The mature hormone, which is located at the carboxyl terminus of the precursor, is preceded by a single pair of basic amino acids. We are studying preproSRIF to investigate intracellular sorting, proteolytic processing, and storage of peptide hormone precursors in the secretory pathway. We used a retroviral expression vector to achieve the high levels of precursor synthesis which are necessary for detailed characterization of processing intermediates and mature somatostatin. Recombinant retroviruses containing RNA transcripts encoding anglerfish preproSRIF I were used to infect rat pituitary GH3 cells which secrete growth hormone and prolactin, neither of which are substrates for endoproteolytic cleavage. In these cells preproSRIF was accurately processed to the mature hormone with an efficiency of approximately 75%. Of the newly synthesized mature SRIF, 55% was sorted into the regulated secretory pathway and released in response to the secretagogue 8-Br-cAMP. The remaining 45% of mature SRIF and residual unprocessed precursor was rapidly secreted. In contrast to SRIF, only 5% of newly synthesized endogenous growth hormone was stored intracellularly, whereas 95% was sorted to the constitutive pathway and secreted rapidly with kinetics identical to proSRIF. Our results show that proSRIF processing is not necessarily dependent on a specific protease found only in SRIF-producing cells and suggest that proteolytic cleavage is not restricted to cells that process endogenous hormones. Moreover, these results demonstrate that GH3 cells have the capacity to discriminate between endogenous and foreign hormones and target the foreign molecule significantly more efficiently to the regulated secretory pathway.
生长抑素（SRIF）是一种由14个氨基酸组成的肽类激素，它作为一个更大的前体——前生长抑素原（preproSRIF）的一部分被合成，前生长抑素原由一个信号肽和一个80 - 90个氨基酸的前体区域组成。成熟激素位于前体的羧基末端，其前面有一对碱性氨基酸。我们正在研究前生长抑素原，以探讨肽类激素前体在分泌途径中的细胞内分选、蛋白水解加工和储存。我们使用逆转录病毒表达载体来实现高水平的前体合成，这对于详细表征加工中间体和成熟生长抑素是必要的。含有编码琵琶鱼前生长抑素原I的RNA转录本的重组逆转录病毒被用于感染大鼠垂体GH3细胞，该细胞分泌生长激素和催乳素，这两种激素都不是内蛋白水解切割的底物。在这些细胞中，前生长抑素原被准确加工成成熟激素，效率约为75%。新合成的成熟SRIF中，55%被分选到调节性分泌途径，并在促分泌剂8 - Br - cAMP的作用下释放。其余45%的成熟SRIF和未加工的残留前体则迅速分泌。与SRIF相反，新合成的内源性生长激素只有5%被细胞内储存，而95%被分选到组成性途径并迅速分泌，其动力学与前生长抑素原相同。我们的结果表明，前生长抑素原的加工不一定依赖于仅在产生SRIF的细胞中发现的特定蛋白酶，这表明蛋白水解切割并不局限于加工内源性激素的细胞。此外，这些结果表明GH3细胞有能力区分内源性和外源性激素，并将外源性分子更有效地靶向调节性分泌途径。
