Yang B, Hall C L, Yang B L, Savani R C, Turley E A
Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Canada.
J Cell Biochem. 1994 Dec;56(4):455-68. doi: 10.1002/jcb.240560406.
We have previously reported that the hyaluronan (HA) receptor RHAMM (Receptor for HA Mediated Motility) [Turley et al., 1991] contains two HA binding motifs located within a 35 amino acid region of its C-terminus end [Yang et al., 1993] and that HA stimulation of the motility of ras-transformed fibroblasts is mediated via its interaction with RHAMM. Here we show that RHAMM also contains binding sites for heparin (HP) and that interaction of HP with these sites can regulate the locomotion of ras-transformed fibroblasts. At low concentrations (0.01 mg/ml), HP inhibited HA-induced locomotion of ras-transformed cells in a manner independent of RHAMM. At higher, but still physiological concentrations (0.1 mg/ml), HP alone stimulated cell locomotion and this stimulation appeared to be RHAMM-dependent as it was blocked by anti-RHAMM antibodies. Other related glycosaminoglycans such as chondroitin sulfate and dermatin sulfate had no effect on cell motility. In ligand blotting assays, GST-RHAMM fusion protein was shown to bind biotin-labelled HP and this binding was displaceable with unlabelled HP. In similar ligand binding analyses conducted with truncations of RHAMM fusion protein, the HP binding region was found to be localized in the same 35 amino acid segment of RHAMM that contains the two HA binding domains. Synthetic peptides corresponding to these HA binding domains were retained on and bound effectively to an HP-Sepharose affinity column. Fusion proteins generated by linkage of these peptides to the non-HP binding amino terminus of RHAMM conferred HP binding capacity to the genetically engineered proteins. Conversely, deletion of the HA binding domains of RHAMM resulted in fusion proteins devoid of HP binding activity. The relative affinities of RHAMM for HA and HP, as determined by competition and transblot assays as well as quantification of binding at various salt concentrations, indicated that RHAMM had lower affinity for HP than that for HA. These results demonstrate the existence of a new HP binding motif that has biological relevance to cell locomotion.
我们之前报道过，透明质酸（HA）受体RHAMM（HA介导的运动受体）[特利等人，1991年]在其C末端的一个35个氨基酸区域内包含两个HA结合基序[杨等人，1993年]，并且HA对ras转化的成纤维细胞运动的刺激是通过其与RHAMM的相互作用介导的。在此我们表明，RHAMM还包含肝素（HP）结合位点，并且HP与这些位点的相互作用可调节ras转化的成纤维细胞的运动。在低浓度（0.01毫克/毫升）时，HP以独立于RHAMM的方式抑制HA诱导的ras转化细胞的运动。在较高但仍为生理浓度（0.1毫克/毫升）时，单独的HP刺激细胞运动，并且这种刺激似乎依赖于RHAMM，因为它被抗RHAMM抗体阻断。其他相关的糖胺聚糖，如硫酸软骨素和硫酸皮肤素，对细胞运动没有影响。在配体印迹分析中，GST-RHAMM融合蛋白显示与生物素标记的HP结合，并且这种结合可被未标记的HP取代。在用RHAMM融合蛋白截短体进行的类似配体结合分析中，发现HP结合区域位于RHAMM的同一35个氨基酸片段中，该片段包含两个HA结合结构域。与这些HA结合结构域相对应的合成肽保留在HP-琼脂糖亲和柱上并与之有效结合。通过将这些肽与RHAMM的非HP结合氨基末端连接产生的融合蛋白赋予了基因工程蛋白HP结合能力。相反，RHAMM的HA结合结构域的缺失导致融合蛋白缺乏HP结合活性。通过竞争和转印分析以及在各种盐浓度下结合的定量测定确定，RHAMM对HP的亲和力低于对HA的亲和力。这些结果证明了一种新HP结合基序的存在，其与细胞运动具有生物学相关性。
