Duda T, Goraczniak R, Surgucheva I, Rudnicka-Nawrot M, Gorczyca W A, Palczewski K, Sitaramayya A, Baehr W, Sharma R K
Department of Cell Biology, University of Medicine and Dentistry of New Jersey, Stratford 08084, USA.
Biochemistry. 1996 Jul 2;35(26):8478-82. doi: 10.1021/bi960752z.
Bovine photoreceptor guanylate cyclase (ROS-GC) consists of a single transmembrane polypeptide chain with extracellular and intracellular domains. In contrast to non-photoreceptor guanylate cyclases (GCs) which are activated by hormone peptides, ROS-GC is modulated in low Ca2+ by calmodulin-like Ca(2+)-binding proteins termed GCAPs (guanylate cyclase-activating proteins). In this communication we show that, like the native system, ROS-GC expressed in COS cells is activated 4-6-fold by recombinant GCAP1 at 10 nM Ca2+ and that the reconstituted system is inhibited at physiological levels of Ca2+ (1 microM). A mutant ROS-GC in which the extracellular domain was deleted was stimulated by GCAP1 indistinguishable from native ROS-GC indicating that this domain is not involved in Ca2+ modulation. Deletion of the intracellular kinase-like domain diminished the stimulation by GCAP1, indicating that this domain is at least in part involved in Ca2+ modulation. Replacement of the catalytic domain in a non-photoreceptor GC by the catalytic domain of ROS-GC yielded a chimeric GC that was sensitive to ANF/ATP and to a lesser extent to GCAP1. The results establish that GCAP1 acts at an intracellular domain, suggesting a mechanism of photoreceptor GC stimulation fundamentally distinct from hormone peptide stimulation of other cyclase receptors.
牛光感受器鸟苷酸环化酶（ROS-GC）由一条具有细胞外和细胞内结构域的单跨膜多肽链组成。与由激素肽激活的非光感受器鸟苷酸环化酶（GCs）不同，ROS-GC在低Ca2+浓度下由称为GCAPs（鸟苷酸环化酶激活蛋白）的钙调蛋白样Ca(2+)-结合蛋白调节。在本通讯中，我们表明，与天然系统一样，在COS细胞中表达的ROS-GC在10 nM Ca2+浓度下被重组GCAP1激活4至6倍，并且重构系统在生理水平的Ca2+（1 microM）下受到抑制。一种缺失细胞外结构域的突变型ROS-GC受到GCAP1的刺激，与天然ROS-GC无法区分，表明该结构域不参与Ca2+调节。缺失细胞内激酶样结构域会减弱GCAP1的刺激作用，表明该结构域至少部分参与Ca2+调节。用ROS-GC的催化结构域替换非光感受器GC中的催化结构域，产生了一种嵌合GC，它对ANF/ATP敏感，对GCAP1的敏感性较低。结果表明，GCAP1作用于细胞内结构域，提示光感受器GC刺激机制与其他环化酶受体的激素肽刺激机制有根本区别。
