Gerendasy D D, Herron S R, Wong K K, Watson J B, Sutcliffe J G
Department of Molecular Biology, Scripps Research Institute, La Jolla, CA 92037, USA.
J Mol Neurosci. 1994 Fall;5(3):133-48. doi: 10.1007/BF02736729.
RC3/Neurogranin is a postnatal-onset, forebrain-specific, thyroid hormone-regulated, protein kinase C (PKC) substrate that binds calmodulin (CaM) and accumulates in dendritic spines. We bacterially expressed and purified RC3 and, for comparison, GAP-43/neuromodulin to near homogeneity using relatively simple procedures. We then raised antisera against recombinant RC3 that does not crossreact with GAP-43 and is suitable for immunohistochemical analysis of brain slices. We also constructed over 30 RC3 sequence variants by PCR-mediated, site-directed mutagenesis, and purified four of these to near homogeneity. The elution profiles displayed by RC3 and sequence variants during purification on CaM-Sepharose columns suggest that two different affinity forms of the RC3.CaM complex coexist when Ca2+ is absent and that GAP-43.CaM interactions are far more sensitive to salt than those that occur between recombinant RC3 and CaM. Variant proteins in which serine 36 was changed failed to serve as a substrate for PKC, implicating this as the target residue.
RC3/神经颗粒素是一种出生后开始表达、前脑特异性、受甲状腺激素调节的蛋白激酶C（PKC）底物，它能结合钙调蛋白（CaM）并在树突棘中积累。我们通过细菌表达并纯化了RC3，为作比较，还用相对简单的方法将GAP-43/神经调节蛋白纯化至接近均一状态。然后我们制备了针对重组RC3的抗血清，该抗血清与GAP-43无交叉反应，适用于脑切片的免疫组织化学分析。我们还通过PCR介导的定点诱变构建了30多种RC3序列变体，并将其中4种纯化至接近均一状态。RC3及其序列变体在钙调蛋白琼脂糖柱上纯化时显示的洗脱曲线表明，在无Ca2+时，RC3.CaM复合物存在两种不同的亲和形式，并且GAP-43.CaM相互作用对盐的敏感性远高于重组RC3与CaM之间的相互作用。丝氨酸36发生改变的变体蛋白不能作为PKC的底物，这表明该丝氨酸是作用靶点。
