Mitra A, Song L, Fricker L D
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461.
J Biol Chem. 1994 Aug 5;269(31):19876-81.
Carboxypeptidase E (CPE), a neuropeptide processing enzyme, is present in neuroendocrine tissues in soluble and membrane-associated forms. The membrane-associated forms do not contain a conventional transmembrane-spanning domain; instead, the C-terminal region of CPE has been proposed to form an amphiphilic helix which binds to the membrane. To test this, and to investigate the possible contribution of this C-terminal sequence to the intracellular sorting of CPE into the regulated pathway, the C-terminal region of CPE was attached to albumin and the recombinant proteins expressed in AtT-20 cells. Albumin itself showed little association with membranes under the conditions examined. A construct containing albumin with only 9 residues of CPE, corresponding to a highly charged region immediately preceding the potential amphiphilic helix region, showed generally similar membrane binding and secretion rates as albumin alone. When the C-terminal 51 amino acids of CPE were attached to the C terminus of albumin and the recombinant protein detected with an antisera raised against the C terminus of CPE, virtually all of the protein was membrane-associated. This finding suggests that the C-terminal region of CPE functions as a membrane anchor. The secretion of albumin with the C-terminal region of CPE was stimulated by a phorbol ester and by forskolin, although the magnitude of the stimulation was smaller than the effect of these compounds on the secretion of CPE. These results imply that the C-terminal region of CPE contains the membrane anchor and contributes to the sorting of this protein into the regulated pathway.
羧肽酶E（CPE）是一种神经肽加工酶，以可溶性和膜相关形式存在于神经内分泌组织中。膜相关形式不包含传统的跨膜结构域；相反，有人提出CPE的C末端区域会形成一个两亲性螺旋，该螺旋与膜结合。为了验证这一点，并研究该C末端序列对CPE细胞内分选进入调节途径的可能贡献，将CPE的C末端区域连接到白蛋白上，并在AtT-20细胞中表达重组蛋白。在检测的条件下，白蛋白本身与膜的结合很少。一种构建体包含仅带有9个CPE残基的白蛋白，这9个残基对应于潜在两亲性螺旋区域之前的一个高电荷区域，其膜结合和分泌速率与单独的白蛋白大致相似。当将CPE的C末端51个氨基酸连接到白蛋白的C末端，并使用针对CPE C末端产生的抗血清检测重组蛋白时，几乎所有蛋白质都与膜相关。这一发现表明CPE的C末端区域起到膜锚定的作用。带有CPE C末端区域的白蛋白的分泌受到佛波酯和福斯高林的刺激，尽管刺激的程度小于这些化合物对CPE分泌的影响。这些结果表明CPE的C末端区域包含膜锚定，并有助于将该蛋白质分选进入调节途径。
