Department of Chemistry, Lehigh University, 6 E. Packer Ave, Bethlehem, Pennsylvania 18015, USA.
Biochemistry. 2012 May 8;51(18):3911-8. doi: 10.1021/bi3001853. Epub 2012 Apr 25.
Caveolin-1 is the most important protein found in caveolae, which are cell surface invaginations of the plasma membrane that act as signaling platforms. A single point mutation in the transmembrane domain of caveolin-1 (proline 132 to leucine) has deleterious effects on caveolae formation in vivo and has been implicated in various disease states, particularly aggressive breast cancers. Using a combination of gel filtration chromatography and analytical ultracentrifugation, we found that a fully functional construct of caveolin-1 (Cav1(62-178)) was a monomer in dodecylphosphocholine micelles. In contrast, the P132L mutant of Cav1(62-178) was dimeric. To explore the dimerization of the P132L mutant further, various truncated constructs (Cav1(82-178), Cav1(96-178), Cav1(62-136), Cav1(82-136), Cav1(96-136)) were prepared which revealed that oligomerization occurs in the transmembrane domain (residues 96-136) of caveolin-1. To characterize the mutant structurally, solution-state NMR experiments in lyso-myristoylphosphatidylglycerol were undertaken of the Cav1(96-136) P132L mutant. Chemical shift analysis revealed that, compared to the wild-type, helix 2 in the transmembrane domain was lengthened by four residues (wild-type, residues 111-129; mutant, residues 111-133), which corresponds to an extra turn in helix 2 of the mutant. Lastly, point mutations at position 132 of Cav1(62-178) (P132A, P132I, P132V, P132G, P132W, P132F) revealed that no other hydrophobic amino acid can preserve the monomeric state of Cav1(62-178), which indicates that proline 132 is critical in supporting proper caveolin-1 behavior.
窖蛋白-1 是质膜凹陷的小窝（ caveolae ）中最重要的蛋白质，小窝作为信号平台。窖蛋白-1 的跨膜结构域中的单个点突变（脯氨酸 132 突变为亮氨酸）对体内小窝的形成有有害影响，并与各种疾病状态有关，特别是侵袭性乳腺癌。通过凝胶过滤色谱和分析超速离心的组合，我们发现具有完整功能的窖蛋白-1 结构域（ Cav1(62-178) ）在十二烷基磷酸胆碱胶束中是单体。相比之下， Cav1(62-178) 的 P132L 突变体是二聚体。为了进一步探索 P132L 突变体的二聚化，制备了各种截断的构建体（ Cav1(82-178) 、 Cav1(96-178) 、 Cav1(62-136) 、 Cav1(82-136) 、 Cav1(96-136) ），结果表明寡聚化发生在窖蛋白-1 的跨膜结构域（残基 96-136 ）中。为了对突变体进行结构表征，在溶酶体-myristoylphosphatidylglycerol 中进行了 Cav1(96-136) P132L 突变体的溶液态 NMR 实验。化学位移分析表明，与野生型相比，跨膜结构域中的螺旋 2 延长了四个残基（野生型，残基 111-129 ；突变体，残基 111-133 ），相当于突变体中螺旋 2 的额外一转。最后，在 Cav1(62-178) 的位置 132 进行点突变（ P132A 、 P132I 、 P132V 、 P132G 、 P132W 、 P132F ）表明，没有其他疏水性氨基酸可以保持 Cav1(62-178) 的单体状态，这表明脯氨酸 132 对支持窖蛋白-1 的正常行为至关重要。
