Institut de Biologie du Développement de Marseille-Luminy, Aix-Marseille Université CNRS 6216, Marseille, France.
PLoS One. 2012;7(2):e32216. doi: 10.1371/journal.pone.0032216. Epub 2012 Feb 22.
Cooperation between receptors allows integrated intracellular signaling leading to appropriate physiological responses. The Neural Cell Adhesion Molecule (NCAM) has three main isoforms of 120, 140 and 180 kDa, with adhesive and signaling properties, but their respective functions remains to be fully identified. Here we show that the human NCAM180 intracellular domain is a novel interactor of the human guanosine exchange factor (GEF) Ric8A using the yeast two hybrid system and immunoprecipitation. Furthermore, NCAM, Ric8A and G(αs) form a tripartite complex. Colocalization experiments by confocal microscopy revealed that human NCAM180 specifically induces the recruitment of Ric8A to the membrane. In addition, using an in vitro recombinant system, and in vivo by comparing NCAM knock-out mouse brain to NCAM heterozygous and wild type brains, we show that NCAM expression dose dependently regulates Ric8A redistribution in detergent resistent membrane microdomains (DRM). Previous studies have demonstrated essential roles for Ric8 in G(α) protein activity at G protein coupled receptors (GPCR), during neurotransmitter release and for asymmetric cell division. We observed that inhibition of Ric8A by siRNA or its overexpression, decreases or increases respectively, cAMP production following β-adrenergic receptor stimulation. Furthermore, in human HEK293T recombinant cells, NCAM180 potentiates the G(αs) coupled β-adrenergic receptor response, in a Ric8A dependent manner, whereas NCAM120 or NCAM140 do not. Finally, in mouse hippocampal neurons expressing endogenously NCAM, NCAM is required for the agonist isoproterenol to induce cAMP production, and this requirement depends on Ric8A. These data illustrate a functional crosstalk between a GPCR and an IgCAM in the nervous system.
受体间的合作允许整合细胞内信号传导，从而导致适当的生理反应。神经细胞黏附分子（NCAM）有三种主要的 120、140 和 180 kDa 同工型，具有黏附和信号转导特性，但它们各自的功能仍有待充分确定。在这里，我们使用酵母双杂交系统和免疫沉淀法显示人 NCAM180 细胞内结构域是人类鸟嘌呤核苷酸交换因子（GEF）Ric8A 的一种新型相互作用蛋白。此外，NCAM、Ric8A 和 G(αs) 形成一个三分体复合物。通过共聚焦显微镜的共定位实验显示，人 NCAM180 特异性诱导 Ric8A 向膜募集。此外，使用体外重组系统，以及通过比较 NCAM 敲除小鼠脑与 NCAM 杂合和野生型脑，我们表明 NCAM 表达剂量依赖性地调节 Ric8A 在去污剂抗性膜微区（DRM）中的重分布。先前的研究表明，Ric8 在 G 蛋白偶联受体（GPCR）中的 G 蛋白活性、神经递质释放和不对称细胞分裂中起着至关重要的作用。我们观察到，Ric8A 的 siRNA 抑制或过表达分别减少或增加β-肾上腺素能受体刺激后的 cAMP 产生。此外，在人 HEK293T 重组细胞中，NCAM180 以 Ric8A 依赖的方式增强 G(αs) 偶联的β-肾上腺素能受体反应，而 NCAM120 或 NCAM140 则没有。最后，在表达内源性 NCAM 的小鼠海马神经元中，NCAM 是激动剂异丙肾上腺素诱导 cAMP 产生所必需的，并且这种需求依赖于 Ric8A。这些数据说明了神经系统中 GPCR 和 IgCAM 之间的功能串扰。
