Singleton Patrick A, Bourguignon Lilly Y W
Department of Medicine, University of California San Francisco, Endocrine Unit (111N), San Francisco VA Medical Center, 94121, USA.
Cell Motil Cytoskeleton. 2002 Dec;53(4):293-316. doi: 10.1002/cm.10078.
Aortic endothelial cells (GM7372A) express a major cell adhesion molecule, CD44v10, which binds the extracellular matrix component, hyaluronan (HA), at its external domain and interacts with various signaling molecules at its cytoplasmic domain. In this study, we have determined that CD44v10 and Rho-Kinase (ROK) are physically associated as a complex in vivo. Using a recombinant fragment of ROK (in particular, the pleckstrin homology [PH] domain) and in vitro binding assays, we have detected a specific binding interaction between the PH domain of ROK and the cytoplasmic domain of CD44. Scatchard plot analysis indicates that there is a single high-affinity CD44 binding site in the PH domain of ROK with an apparent dissociation constant (Kd) of 1.76 nM, which is comparable to CD44 binding (Kd approximately 1.56 nM) to intact ROK. These findings suggest that the PH domain is the primary ROK binding region for CD44. Furthermore, HA binding to GM7372A cells promotes RhoA-mediated ROK activity, which, in turn, increases phosphorylation of three different inositol 1, 4, 5-trisphosphate receptors (IP(3)Rs) [in particular, subtype 1 (IP(3)R1), and to a lesser extent subtype 2 (IP(3)R2) and subtype 3 (IP(3)R3)] all known as IP(3)-gated Ca(2+) channels. The phosphorylated IP(3)R1 (but not IP(3)R2 or IP(3)R3) is enhanced in its binding to IP(3) which subsequently stimulates IP(3)-mediated Ca(2+) flux. Transfection of the endothelial cells with ROK's PH cDNA significantly reduces ROK association with CD44v10, and effectively inhibits ROK-mediated phosphorylation of IP(3)Rs and IP(3)R-mediated Ca(2+) flux in vitro. The PH domain of ROK also functions as a dominant-negative mutant in vivo to block HA-dependent, CD44v10-specific intracellular Ca(2+) mobilization and endothelial cell migration. Taken together, we believe that CD44v10 interaction with ROK plays a pivotal role in IP(3)R-mediated Ca(2+) signaling during HA-mediated endothelial cell migration.
主动脉内皮细胞（GM7372A）表达一种主要的细胞粘附分子CD44v10，其在细胞外结构域与细胞外基质成分透明质酸（HA）结合，并在细胞质结构域与各种信号分子相互作用。在本研究中，我们确定CD44v10与Rho激酶（ROK）在体内以复合物形式物理结合。使用ROK的重组片段（特别是普列克底物蛋白同源[PH]结构域）和体外结合试验，我们检测到ROK的PH结构域与CD44的细胞质结构域之间存在特异性结合相互作用。Scatchard作图分析表明，ROK的PH结构域中有一个单一的高亲和力CD44结合位点，其表观解离常数（Kd）为1.76 nM，这与CD44与完整ROK的结合（Kd约为1.56 nM）相当。这些发现表明，PH结构域是ROK与CD44的主要结合区域。此外，HA与GM7372A细胞的结合促进RhoA介导的ROK活性，进而增加三种不同的肌醇1,4,5-三磷酸受体（IP(3)Rs）[特别是亚型1（IP(3)R1），以及程度较轻的亚型2（IP(3)R2）和亚型3（IP(3)R3）]的磷酸化，这些受体均为IP(3)门控Ca(2+)通道。磷酸化的IP(3)R1（而非IP(3)R2或IP(3)R3）与IP(3)的结合增强，随后刺激IP(3)介导的Ca(2+)内流。用ROK的PH cDNA转染内皮细胞可显著降低ROK与CD44v10的结合，并有效抑制体外ROK介导的IP(3)Rs磷酸化和IP(3)R介导的Ca(2+)内流。ROK的PH结构域在体内也作为显性负性突变体发挥作用，以阻断HA依赖性、CD44v10特异性的细胞内Ca(2+)动员和内皮细胞迁移。综上所述，我们认为CD44v10与ROK的相互作用在HA介导的内皮细胞迁移过程中IP(3)R介导的Ca(2+)信号传导中起关键作用。
