Brouillet E, Trembleau A, Galanaud D, Volovitch M, Bouillot C, Valenza C, Prochiantz A, Allinquant B
Centre National de la Recherche Scientifique, Unité de Recherche Associée 1414, Ecole Normale Supérieure, 75230 Paris Cedex 05, France.
J Neurosci. 1999 Mar 1;19(5):1717-27. doi: 10.1523/JNEUROSCI.19-05-01717.1999.
The function of the beta-amyloid protein precursor (betaAPP), a transmembrane molecule involved in Alzheimer pathologies, is poorly understood. We recently reported the presence of a fraction of betaAPP in cholesterol and sphingoglycolipid-enriched microdomains (CSEM), a caveolae-like compartment specialized in signal transduction. To investigate whether betaAPP actually interferes with cell signaling, we reexamined the interaction between betaAPP and Go GTPase. In strong contrast with results obtained with reconstituted phospholipid vesicles (Okamoto et al., 1995), we find that incubating total neuronal membranes with 22C11, an antibody that recognizes an N-terminal betaAPP epitope, reduces high-affinity Go GTPase activity. This inhibition is specific of Galphao and is reproduced, in the absence of 22C11, by the addition of the betaAPP C-terminal domain but not by two distinct mutated betaAPP C-terminal domains that do not bind Galphao. This inhibition of Galphao GTPase activity by either 22C11 or wild-type betaAPP cytoplasmic domain suggests that intracellular interactions between betaAPP and Galphao could be regulated by extracellular signals. To verify whether this interaction is preserved in CSEM, we first used biochemical, immunocytochemical, and ultrastructural techniques to unambiguously confirm the colocalization of Galphao and betaAPP in CSEM. We show that inhibition of basal Galphao GTPase activity also occurs within CSEM and correlates with the coimmunoprecipitation of Galphao and betaAPP. The regulation of Galphao GTPase activity by betaAPP in a compartment specialized in signaling may have important consequences for our understanding of the physiopathological functions of betaAPP.
β-淀粉样蛋白前体（βAPP）是一种参与阿尔茨海默病病理过程的跨膜分子，其功能尚不清楚。我们最近报道了在富含胆固醇和鞘糖脂的微结构域（CSEM）中存在一部分βAPP，CSEM是一种类似小窝的、专门参与信号转导的区室。为了研究βAPP是否真的干扰细胞信号传导，我们重新检查了βAPP与Go GTP酶之间的相互作用。与用重组磷脂囊泡获得的结果（冈本等人，1995年）形成强烈对比的是，我们发现用识别βAPP N端表位的抗体22C11孵育总神经细胞膜会降低高亲和力Go GTP酶活性。这种抑制作用是Gαo特异性的，并且在没有22C11的情况下，通过添加βAPP C端结构域可以重现，但添加两个不与Gαo结合的不同突变βAPP C端结构域则不会重现。22C11或野生型βAPP胞质结构域对Gαo GTP酶活性的这种抑制作用表明，βAPP与Gαo之间的细胞内相互作用可能受细胞外信号调节。为了验证这种相互作用在CSEM中是否存在，我们首先使用生化、免疫细胞化学和超微结构技术明确证实了Gαo和βAPP在CSEM中的共定位。我们表明，CSEM内也会发生对基础Gαo GTP酶活性的抑制，并且这与Gαo和βAPP的共免疫沉淀相关。βAPP在专门参与信号传导的区室中对Gαo GTP酶活性的调节可能对我们理解βAPP的生理病理功能具有重要意义。
