Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico, USA.
Nat Struct Mol Biol. 2011 Oct 23;18(11):1244-9. doi: 10.1038/nsmb.2135.
The extent to which ligand occupancy and dimerization contribute to erbB1 signaling is controversial. To examine this, we used two-color quantum-dot tracking for visualization of the homodimerization of human erbB1 and quantification of the dimer off-rate (k(off)) on living cells. Kinetic parameters were extracted using a three-state hidden Markov model to identify transition rates between free, co-confined and dimerized states. We report that dimers composed of two ligand-bound receptors are long-lived and their k(off) is independent of kinase activity. By comparison, unliganded dimers have a more than four times faster k(off). Transient co-confinement of receptors promotes repeated encounters and enhances dimer formation. Mobility decreases more than six times when ligand-bound receptors dimerize. Blockade of erbB1 kinase activity or disruption of actin networks results in faster diffusion of receptor dimers. These results implicate both signal propagation and the cortical cytoskeleton in reduced mobility of signaling-competent erbB1 dimers.
配体占据和二聚化在多大程度上促进 erbB1 信号转导一直存在争议。为了研究这一点，我们使用双色量子点追踪技术可视化人 erbB1 的同源二聚化，并定量测定活细胞中二聚体的离解速率 (k(off))。我们使用三状态隐马尔可夫模型提取动力学参数，以识别自由、共限制和二聚化状态之间的转换速率。我们报告说，由两个配体结合受体组成的二聚体是长寿命的，其 k(off)与激酶活性无关。相比之下，未配体结合的二聚体的 k(off)快四倍多。受体的瞬时共限制促进了反复的接触并增强了二聚体的形成。当配体结合的受体二聚化时，流动性降低了六倍以上。阻断 erbB1 激酶活性或破坏肌动蛋白网络会导致受体二聚体的扩散更快。这些结果表明信号转导和皮质细胞骨架都参与了信号传导有效 erbB1 二聚体的流动性降低。
