Estrada Kristine, Fisher Carl, Blacklow Stephen C
Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Biochemistry. 2008 Feb 12;47(6):1532-9. doi: 10.1021/bi702076y. Epub 2008 Jan 5.
The receptor-associated protein (RAP) functions as an escort protein for receptors of the low-density lipoprotein receptor (LDLR) family by preventing premature intracellular binding of ligands and assisting with delivery of mature receptors to the cell surface. The modulation of affinity by pH is believed to play an important role in the escort function of RAP, because RAP binds tightly to proteins of the LDLR family at near-neutral pH early in the secretory pathway where its high affinity precludes premature binding of ligands but then dissociates from bound receptors at the lower pH of the Golgi compartment. The third domain of RAP (RAP-D3), which forms a three-helix bundle, is sufficient to reconstitute the escort activity. Here, we test the hypothesis that low-pH induced unfolding of the RAP-D3 helical bundle facilitates dissociation of RAP-receptor complexes. First, variants of RAP-D3 resistant to low pH-induced unfolding were constructed by replacing interior histidine residues with phenylalanines. In contrast to native RAP-D3, which exhibits an unfolding pKa of 6.3 and a Tm of 42 degrees C, the most hyperstable variant of RAP-D3, in which four histidine residues are replaced with phenylalanine, has an unfolding pKa of 4.8, and a Tm of 58 degrees C. The phenylalanine substitutions in RAP-D3 confer increased stability to pH-induced dissociation of complexes formed between RAP-D3 and a two-repeat fragment of the LDLR (LA3-4). When introduced into full-length RAP, the four mutations that confer hyperstability on RAP-D3 interfere with transport of endogenous LRP-1 to the cell surface in a dominant negative fashion under conditions where expression of normal RAP has no effect on LRP-1 transport. Our studies support a model in which low pH-dependent unfolding of RAP-D3 facilitates dissociation of RAP from the LA repeats of LDLR family proteins in the mildly acidic pH of the Golgi.
受体相关蛋白（RAP）作为低密度脂蛋白受体（LDLR）家族受体的护送蛋白，通过防止配体过早地在细胞内结合，并协助将成熟受体递送至细胞表面来发挥作用。pH对亲和力的调节被认为在RAP的护送功能中起重要作用，因为RAP在分泌途径早期接近中性pH时与LDLR家族的蛋白紧密结合，此时其高亲和力可防止配体过早结合，但随后在高尔基体区室较低的pH下与结合的受体解离。RAP的第三个结构域（RAP-D3）形成一个三螺旋束，足以重建护送活性。在此，我们检验了这样一个假说，即低pH诱导的RAP-D3螺旋束解折叠促进了RAP-受体复合物的解离。首先，通过用苯丙氨酸取代内部组氨酸残基构建了对低pH诱导解折叠具有抗性的RAP-D3变体。与天然RAP-D3（其解折叠pKa为6.3，熔解温度为42℃）不同，RAP-D3最稳定的变体（其中四个组氨酸残基被苯丙氨酸取代）的解折叠pKa为4.8，熔解温度为58℃。RAP-D3中的苯丙氨酸取代增加了对pH诱导的RAP-D3与LDLR的两个重复片段（LA3-4）形成的复合物解离的稳定性。当引入全长RAP中时，赋予RAP-D3超稳定性的四个突变在正常RAP表达对LRP-1转运无影响的条件下，以显性负性方式干扰内源性LRP-1向细胞表面的转运。我们的研究支持这样一个模型，即在高尔基体轻度酸性pH条件下，RAP-D3的低pH依赖性解折叠促进了RAP与LDLR家族蛋白的LA重复序列的解离。
