Cali J J, Parekh R S, Krupinski J
Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822-2610, USA.
J Biol Chem. 1996 Jan 12;271(2):1089-95. doi: 10.1074/jbc.271.2.1089.
Three alternatively spliced type VIII adenylyl cyclase messages have been identified by cDNA cloning and amplification from rat brain cDNA. Type VIII-A was previously referred to simply as type VIII (Cali, J. J., Zwaagstra, J. C., Mons, N., Cooper, D. M. F., and Krupinski, J. (1994) J. Biol. Chem. 269, 12190-12195). The types VIII-B and -C cDNAs differ from that of type VIII-A by deletion of 90 and 198 base pair exons, respectively, which encode a 30-amino acid extracellular domain with two consensus sites for N-linked glycosylation and a 66-amino acid cytoplasmic domain. Stable expression of types VIII-A, -B, and -C cDNAs in human embryonal kidney 293 (HEK-293) cells leads to the appearance of novel proteins, which are recognized by type VIII-specific antibodies and which co-migrate with immunoreactive species detected on immunoblots of rat brain membranes. Types VIII-A and -C are modified by N-linked glycosylation, while type VIII-B is insensitive to treatment with N-glycosidase F. An influx of extracellular Ca2+ stimulates cAMP accumulation in HEK-293 cells stably expressing type VIII-A, -B, or -C, but not in control cells. Adenylyl cyclase activity of each of the variants is stimulated by Ca2+/calmodulin and the EC50 for activation of type VIII-C is one fourth of that for either type VIII-A or -B. Type VIII-C also has a distinct Km for substrate, which is approximately 4-12-fold higher than that for types VIII-A or -B depending on whether Mn2+ or Mg2+ is the counterion for ATP. The differences in the structural and enzymatic properties of these three variants are discussed.
通过从大鼠脑cDNA进行cDNA克隆和扩增，已鉴定出三种选择性剪接的VIII型腺苷酸环化酶信使。VIII - A型以前简称为VIII型（卡利，J. J.，兹瓦格斯特拉，J. C.，蒙斯，N.，库珀，D. M. F.，和克鲁平斯基，J.（1994年）《生物化学杂志》269，12190 - 12195）。VIII - B型和 - C型cDNA分别与VIII - A型cDNA不同，它们缺失了90和198个碱基对外显子，这些外显子编码一个具有两个N - 糖基化共有位点的30个氨基酸的细胞外结构域和一个66个氨基酸的细胞质结构域。VIII - A型、 - B型和 - C型cDNA在人胚肾293（HEK - 293）细胞中的稳定表达导致出现新的蛋白质，这些蛋白质可被VIII型特异性抗体识别，并且与在大鼠脑膜免疫印迹上检测到的免疫反应性物质共迁移。VIII - A型和 - C型被N - 糖基化修饰，而VIII - B型对N - 糖苷酶F处理不敏感。细胞外Ca2 +的流入刺激稳定表达VIII - A型、 - B型或 - C型的HEK - 293细胞中的cAMP积累，但不刺激对照细胞。每种变体的腺苷酸环化酶活性都受到Ca2 + /钙调蛋白的刺激，VIII - C型激活的EC50是VIII - A型或 - B型的四分之一。VIII - C型对底物也有不同的Km值，根据Mn2 +还是Mg2 +作为ATP的抗衡离子，其比VIII - A型或 - B型高约4 - 12倍。讨论了这三种变体在结构和酶学性质上的差异。
