Thorne B A, Caton L W, Thomas G
Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201.
J Biol Chem. 1989 Feb 25;264(6):3545-52.
Proopiomelanocortin (POMC) is a neuroendocrine precursor protein which is processed at paired basic amino acids in a tissue-specific manner. To study this phenomenon, a vaccinia virus recombinant, which directs the synthesis of mouse POMC (VV:mPOMC) was constructed and used to infect epithelial (BSC-40) and endocrine (Rin m5F) cell lines. Bona fide mPOMC was produced in both cell types and beta-endorphin immunoreactivity was secreted in a nonregulated manner from BSC-40 cells and in a regulated manner from Rin m5F cells. Although the precursor was not cleaved to smaller beta-MSH or beta-endorphin immunoreactive peptides in BSC-40 cell extracts, Rin m5F cells produced primarily authentic gamma-lipotropin and des-acetyl beta-endorphin. Furthermore, production of these peptides was restricted to the regulated secretory pathway in Rin m5F cells. Site-directed mutagenesis was then used to change the inefficiently recognized Lys-Lys potential cleavage site near the carboxyl terminus of beta-endorphin to Lys-Arg. Expression of the mutant precursor in Rin m5F cells resulted in the synthesis of both des-acetyl beta-endorphin and beta-endorphin.
阿黑皮素原（POMC）是一种神经内分泌前体蛋白，它以组织特异性方式在成对的碱性氨基酸处进行加工。为了研究这一现象，构建了一种指导小鼠POMC合成的痘苗病毒重组体（VV:mPOMC），并用于感染上皮细胞系（BSC-40）和内分泌细胞系（Rin m5F）。在这两种细胞类型中都产生了真正的mPOMC，β-内啡肽免疫反应性以非调节方式从BSC-40细胞分泌，而从Rin m5F细胞以调节方式分泌。虽然在BSC-40细胞提取物中前体未被切割成较小的β-MSH或β-内啡肽免疫反应性肽，但Rin m5F细胞主要产生真正的γ-促脂素和去乙酰基β-内啡肽。此外，这些肽的产生仅限于Rin m5F细胞中的调节性分泌途径。然后使用定点诱变将β-内啡肽羧基末端附近识别效率低的Lys-Lys潜在切割位点改变为Lys-Arg。突变前体在Rin m5F细胞中的表达导致了去乙酰基β-内啡肽和β-内啡肽的合成。
