CLONING, SEQUENCING, AND EXPRESSION OF A 24-KDA CA2+-BINDING PROTEIN ACTIVATING PHOTORECEPTOR GUANYLYL CYCLASE
CLONING, SEQUENCING, AND EXPRESSION OF A 24-KDA CA2+-BINDING PROTEIN ACTIVATING PHOTORECEPTOR GUANYLYL CYCLASE
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DOI：
10.1074/jbc.270.42.25200
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发表时间：
1995-10-20
影响因子：
4.8
通讯作者：
HURLEY, JB
中科院分区：
文献类型：
--
作者：
DIZHOOR, AM;OLSHEVSKAYA, EV;HURLEY, JB
两种脊椎动物光感受器特异性膜guanyl环化酶RetGC-1和RetGC-2被可溶性24kda视网膜蛋白p24以Ca2+敏感方式激活(Dizhoor, a . M., Lowe， D. G., Olshevskaya， E. V., Laura， R. P., and Hurley， J. B. (1994) Neuron 12, 1345-1352；罗威，D. G., Dizhoor， A. M.，刘，K.，顾，O., Laura， R., Lu， L.，和Hurley， J. B. (1995) Proc. Natl。学会科学。美国法典92,5535-5539)。牛p24的初级结构已通过肽段测序和cDNA得到。p24是一种新的ef -手型Ca2+结合蛋白，与另一种冠基环化酶激活蛋白GCAP相关但不相同（Palczewski， K., Subbaraya， I., Gorczyca， W. a ., Helekar， B. S., Ruiz， C. C., Ohguro， H. Huang， J., Zhao， X., Crabb， J. W., Johnson， R. S., Walsh， K. a ., Gray-Keller， M. P., Detwiler， P. B., Baehr， W. (1994) Neuron 13, 395-404）和Ca2+结合蛋白恢复家族的其他成员。抗截断融合蛋白和抗p24特异性合成肽的抗体在免疫印迹上特异性识别视网膜p24。这两种抗体通过纯化的p24抑制光感受器膜观基环化酶的激活。P24仅存在于视网膜中，并与外节膜结合。免疫细胞化学分析显示它存在于杆状光感受器细胞中。固定化抗体柱用于从热处理的视网膜提取物中纯化p24。纯化后的p24在sds -聚丙烯酰胺凝胶电泳上表现为未被GCAP污染的均质蛋白，在体外以亚微摩尔浓度激活光感受器鸟酰环化酶。Ca2+抑制这种激活的EC（50）在200 nM附近，Hill系数为1.7。重组p24在293细胞中表达，能有效刺激光感受器鸟苷环化酶。这些发现表明p24，像GCAP一样，赋予Ca2+对光感受器膜guanyyl环化酶的敏感性。我们建议将p24称为GCAP-2，将GCAP称为GCAP-1。
Two vertebrate photoreceptor-specific membrane guanylyl cyclases, RetGC-1 and RetGC-2, are activated by a soluble 24-kDa retinal protein, p24, in a Ca2+-sensitive manner (Dizhoor, A. M., Lowe, D. G., Olshevskaya, E. V., Laura, R. P., and Hurley, J. B. (1994) Neuron 12, 1345-1352; Lowe, D. G., Dizhoor, A. M., Liu, K., Gu, O., Laura, R., Lu, L., and Hurley, J. B. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5535-5539). The primary structure of bovine p24 has been derived from peptide sequencing and from its cDNA. p24 is a new EF-hand-type Ca2+ binding protein, related but not identical to another guanylyl cyclase-activating protein, GCAP (Palczewski, K., Subbaraya, I., Gorczyca, W. A., Helekar, B. S., Ruiz, C. C., Ohguro, H. Huang, J., Zhao, X., Crabb, J. W., Johnson, R. S., Walsh, K. A., Gray-Keller, M. P., Detwiler, P. B., and Baehr, W. (1994) Neuron 13, 395-404) and other members of the recoverin family of Ca2+-binding proteins. Antibodies against a truncated fusion protein and against a p24-specific synthetic peptide specifically recognize retinal p24 on immunoblot. Both antibodies inhibit activation of photoreceptor membrane guanylyl cyclase by purified p24. p24 is found only in retina, and it copurifies with outer segment membranes. Immunocytochemical analysis shows that it is present in rod photoreceptor cells. An immobilized antibody column was used to purify p24 from a heat-treated retinal extract. Purified p24 appears on SDS-polyacrylamide gel electrophoresis as a homogenous protein not contaminated with GCAP, and it activates photoreceptor guanylyl cyclase in vitro at submicromolar concentrations. Ca2+ inhibits this activation with an EC(50) near 200 nM and a Hill coefficient of 1.7. Recombinant p24 expressed in 293 cells effectively stimulates photoreceptor guanylyl cyclase. These findings demonstrate that p24, like GCAP, imparts Ca2+ sensitivity to photoreceptor membrane guanylyl cyclase. We propose that p24 be referred to as GCAP-2 and that GCAP be referred to as GCAP-1.