Department of Pathology, Harvard Medical School, Boston, Massachusetts 02215, USA.
J Biol Chem. 2011 Jul 15;286(28):25171-86. doi: 10.1074/jbc.M111.242362. Epub 2011 May 23.
The normal expression, cell surface localization, and function of the murine high density lipoprotein receptor scavenger receptor class B type I (SR-BI) in hepatocytes in vivo, and thus normal lipoprotein metabolism, depend on its four PDZ domain (PDZ1-PDZ4) containing cytoplasmic adaptor protein PDZK1. Previous studies showed that the C terminus of SR-BI ("target peptide") binds directly to PDZ1 and influences hepatic SR-BI protein expression. Unexpectedly an inactivating mutation in PDZ1 (Tyr(20) → Ala) only partially, rather than completely, suppresses the ability of PDZK1 to control hepatic SR-BI. We used isothermal titration calorimetry to show that PDZ3, but not PDZ2 or PDZ4, can also bind the target peptide (K(d) = 37.0 μm), albeit with ∼10-fold lower affinity than PDZ1. This binding is abrogated by a Tyr(253) → Ala substitution. Comparison of the 1.5-Å resolution crystal structure of PDZ3 with its bound target peptide ((505)QEAKL(509)) to that of peptide-bound PDZ1 indicated fewer target peptide stabilizing atomic interactions (hydrogen bonds and hydrophobic interactions) in PDZ3. A double (Tyr(20) → Ala (PDZ1) + Tyr(253) → Ala (PDZ3)) substitution abrogated all target peptide binding to PDZK1. In vivo hepatic expression of a singly substituted (Tyr(253) → Ala (PDZ3)) PDZK1 transgene (Tg) was able to correct all of the SR-BI-related defects in PDZK1 knock-out mice, whereas the doubly substituted [Tyr(20) → Ala (PDZ1) + Tyr(253) → Ala (PDZ3)]Tg was unable to correct these defects. Thus, we conclude that PDZK1-mediated control of hepatic SR-BI requires direct binding of the SR-BI C terminus to either the PDZ1 or PDZ3 domains, and that binding to both domains simultaneously is not required for PDZK1 control of hepatic SR-BI.
在体内，鼠源高密度脂蛋白受体清道夫受体 B 型 I（SR-BI）在肝细胞中的正常表达、细胞表面定位和功能，以及正常的脂蛋白代谢，都依赖于其含有四个 PDZ 结构域（PDZ1-PDZ4）的细胞质衔接蛋白 PDZK1。先前的研究表明，SR-BI 的 C 末端（“靶肽”）直接与 PDZ1 结合，并影响肝脏 SR-BI 蛋白的表达。出乎意料的是，PDZ1 中的一个失活突变（Tyr（20）→Ala）仅部分而非完全抑制了 PDZK1 控制肝 SR-BI 的能力。我们使用等温滴定量热法表明，PDZ3 而不是 PDZ2 或 PDZ4，也可以与靶肽结合（K(d)=37.0 μm），尽管亲和力比 PDZ1 低约 10 倍。这种结合被 Tyr（253）→Ala 取代所阻断。PDZ3 与其结合的靶肽（(505)QEAKL(509)）的 1.5-Å 分辨率晶体结构与肽结合的 PDZ1 的晶体结构进行比较，表明 PDZ3 中靶肽稳定的原子相互作用（氢键和疏水相互作用）较少。双（Tyr（20）→Ala（PDZ1）+Tyr（253）→Ala（PDZ3））取代消除了 PDZK1 与所有靶肽的结合。在体内，肝表达单取代（Tyr（253）→Ala（PDZ3））PDZK1 转基因（Tg）能够纠正 PDZK1 敲除小鼠中所有与 SR-BI 相关的缺陷，而双取代[Tyr（20）→Ala（PDZ1）+Tyr（253）→Ala（PDZ3）]Tg 不能纠正这些缺陷。因此，我们得出结论，PDZK1 介导的肝 SR-BI 控制需要 SR-BI C 末端直接与 PDZ1 或 PDZ3 结构域结合，并且 PDZK1 控制肝 SR-BI 不需要同时与两个结构域结合。
