Sevarino K A, Stork P
Department of Medicine, Tufts-New England Medical Center, Boston, Massachusetts 02111.
J Biol Chem. 1991 Oct 5;266(28):18507-13.
We have previously detected a sorting signal in the amino-terminal 78 residues of rat preprosomatostatin (rPPSS) that targets the precursor into a regulated secretory pathway or pathways allowing proteolytic maturation (Sevarino, K. A., Stork, P., Ventimiglia, R., Mandel, G., and Goodman, R. H. (1989) Cell 57, 11-19). To further localize this signal, we constructed three rPPSS expression vectors that code for substitutions or mutations spanning that portion of rPPSS implicated in sorting, and the precursors were expressed in RIN 5F cells. Fractionation of the intracellular products revealed that accurate processing to somatostatin-14 (SS-14) was not affected by any of the mutations. Examination of the secreted products showed no reduction in processing efficiency, indicating that none of the mutations blocked sorting from constitutive into regulated secretion. Finally, we examined the response to two separate secretogogues, cAMP and 12-O-tetradecanoylphorbol-13-acetate (TPA). Clones expressing two of the three mutant precursors displayed the same stimulation of SS-14 secretion by exogenously administered cAMP and TPA as cells expressing wild-type rPPSS, indicating that targeting specifically to the secretory pathway, or pathways, responsive to cAMP and TPA was not disrupted. However, cells expressing the mutant precursor containing a substitution of the amino-terminal 34 residues of rPPSS by the amino terminus of the vesicular stomatitis virus G protein displayed greatly reduced stimulation of SS-14 secretion by TPA, with a less than compensatory increase in response to cAMP, when compared to cells expressing wild-type rPPSS. In conjunction with our previous studies with anglerfish preprosomatostatins, we conclude that 1) the sorting signal(s) in rPPSS necessary for cAMP-responsive secretion are redundant and probably reside within both mature peptide regions and extrapeptide regions; 2) two or more distinct regulated secretory pathways utilized by secreted peptides can be demonstrated in transfected endocrine cells and targeting to these pathways can be separately mediated by at least two different types of sorting signals within the neuropeptide precursor itself; and 3) pro-region conformation plays little role in prosomatostatin-processing site recognition.
我们之前在大鼠前促生长抑素原（rPPSS）氨基末端的78个残基中检测到一个分选信号，该信号可将前体靶向到一条或多条受调控的分泌途径，或允许进行蛋白水解成熟的途径（塞瓦里诺，K.A.，斯托克，P.，文蒂米利亚，R.，曼德尔，G.，古德曼，R.H.（1989年）《细胞》57卷，11 - 19页）。为了进一步定位这个信号，我们构建了三个rPPSS表达载体，它们编码跨越rPPSS中与分选相关部分的替换或突变，并且这些前体在RIN 5F细胞中表达。对细胞内产物进行分级分离显示，向生长抑素 - 14（SS - 14）的准确加工不受任何一种突变的影响。对分泌产物的检测表明加工效率没有降低，这表明没有一个突变阻止从组成型分泌到受调控分泌的分选。最后，我们检测了对两种不同促分泌素，即环磷酸腺苷（cAMP）和12 - O - 十四酰佛波醇 - 13 - 乙酸酯（TPA）的反应。表达三种突变前体中两种的克隆对外源性给予的cAMP和TPA刺激SS - 14分泌的反应，与表达野生型rPPSS的细胞相同，这表明特异性靶向对cAMP和TPA有反应的分泌途径没有被破坏。然而，与表达野生型rPPSS的细胞相比，表达将rPPSS氨基末端34个残基替换为水泡性口炎病毒G蛋白氨基末端的突变前体的细胞，TPA刺激SS - 14分泌的作用大大降低，对cAMP的反应增加不足以为其补偿。结合我们之前对安康鱼前促生长抑素原的研究，我们得出以下结论：1）cAMP反应性分泌所需的rPPSS中的分选信号是冗余的，可能存在于成熟肽区域和肽外区域；2）在转染的内分泌细胞中可以证明分泌肽利用两条或更多条不同的受调控分泌途径，并且靶向这些途径可以由神经肽前体本身内至少两种不同类型的分选信号分别介导；3）前体区域构象在促生长抑素加工位点识别中作用很小。
