From the Department of Basic Sciences and the Pennsylvania College of Optometry, Salus University, Elkins Park, Pennsylvania 19027 and.
J Biol Chem. 2014 Apr 4;289(14):10140-54. doi: 10.1074/jbc.M113.540716. Epub 2014 Feb 24.
Retinal guanylyl cyclase (RetGC)-activating proteins (GCAPs) regulate visual photoresponse and trigger congenital retinal diseases in humans, but GCAP interaction with its target enzyme remains obscure. We mapped GCAP1 residues comprising the RetGC1 binding site by mutagenizing the entire surface of GCAP1 and testing the ability of each mutant to bind RetGC1 in a cell-based assay and to activate it in vitro. Mutations that most strongly affected the activation of RetGC1 localized to a distinct patch formed by the surface of non-metal-binding EF-hand 1, the loop and the exiting helix of EF-hand 2, and the entering helix of EF-hand 3. Mutations in the binding patch completely blocked activation of the cyclase without affecting Ca(2+) binding stoichiometry of GCAP1 or its tertiary fold. Exposed residues in the C-terminal portion of GCAP1, including EF-hand 4 and the helix connecting it with the N-terminal lobe of GCAP1, are not critical for activation of the cyclase. GCAP1 mutants that failed to activate RetGC1 in vitro were GFP-tagged and co-expressed in HEK293 cells with mOrange-tagged RetGC1 to test their direct binding in cyto. Most of the GCAP1 mutations introduced into the "binding patch" prevented co-localization with RetGC1, except for Met-26, Lys-85, and Trp-94. With these residues mutated, GCAP1 completely failed to stimulate cyclase activity but still bound RetGC1 and competed with the wild type GCAP1. Thus, RetGC1 activation by GCAP1 involves establishing a tight complex through the binding patch with an additional activation step involving Met-26, Lys-85, and Trp-94.
视网膜鸟苷酸环化酶（RetGC）激活蛋白（GCAPs）调节视觉光反应，并在人类中引发先天性视网膜疾病，但 GCAP 与靶酶的相互作用仍不清楚。我们通过突变 GCAP1 的整个表面，定位包含 RetGC1 结合位点的 GCAP1 残基，并在基于细胞的测定中测试每个突变体与 RetGC1 结合的能力以及在体外激活它的能力，来研究 GCAP1。最强烈影响 RetGC1 激活的突变定位于由非金属结合 EF 手 1 的表面、环和 EF 手 2 的出线螺旋以及 EF 手 3 的进线螺旋形成的独特斑块。结合斑块中的突变完全阻断了环化酶的激活，而不影响 GCAP1 的 Ca(2+)结合计量或其三级折叠。GCAP1 的 C 末端部分暴露的残基，包括 EF 手 4 和连接它与 GCAP1 的 N 末端叶的螺旋，对于环化酶的激活不是关键的。在体外无法激活 RetGC1 的 GCAP1 突变体被 GFP 标记，并与 mOrange 标记的 RetGC1 共表达在 HEK293 细胞中，以在细胞中测试它们的直接结合。除了 Met-26、Lys-85 和 Trp-94 之外，引入 GCAP1 的“结合斑块”中的大多数突变都阻止了与 RetGC1 的共定位。用这些残基突变，GCAP1 完全无法刺激环化酶活性，但仍与 RetGC1 结合，并与野生型 GCAP1 竞争。因此，GCAP1 对 RetGC1 的激活涉及通过结合斑块建立紧密复合物，然后涉及 Met-26、Lys-85 和 Trp-94 的额外激活步骤。
