FACTORS INVOLVED IN B-CAM/LU MEDIATED ADHESION

B-CAM/LU是一种黏附分子，在上皮性癌和镰状细胞病患者的红细胞上表达增加。我们提出的假设是，B-CAM/LU在镰状细胞病中既介导层粘连蛋白的黏附，也介导层粘连蛋白与细胞间的黏附，从而在导致疼痛和器官损伤的血管闭塞过程中发挥重要作用。我们进一步建议看到这些研究的结果，并探索通过开发能够干扰B-CAM/LU与其配体结合的试剂来减少SS RBC与内皮细胞和内皮下基质层粘连蛋白的粘连的治疗方法。首先，我们将使用多种方法来鉴定在B-CAM/LU介导的黏附过程中作为配体的RBC和内皮细胞的表面分子。其次，我们将确定在S红细胞和表达重组形式的B-CAM/LU有核细胞上激活B-CAM/LU黏附功能所涉及的过程。磷酸化和蛋白质-蛋白质相互作用都可能在这一过程中发挥作用。具体地说，我们将根据初步数据研究B-CAM/LU与整合素(特别是α4beta1)和CD44可能的相互作用。我们还将研究B-CAM/LU胞浆结构域中丝氨酸/苏氨酸磷酸化的影响，以及这种作用在SS和正常RBC以及氧合和去氧RBC中是否有不同程度的发生。我们将进一步确定假定的B-CAM/LU的SH3结合基序是否与酪氨酸激酶或其他具有SH3基序的信号分子相关联。第三，为了开发阻断B-CAM/LU依赖的黏附的特异性抑制剂，我们将使用各种黏附实验来研究抗B-CAM/LU单抗以及通过筛选多肽展示噬菌体文库鉴定的可溶性重组B-CAM/LU和抑制肽的作用。总体而言，这些研究将进一步确定B-CAM/LU如何促进粘连和血管闭塞，并将确定其相互作用在治疗环境中可能被取消的途径。

B-CAM/LU is an adhesion molecule whose expression is increased in epithelial cancers and on red cells (RBC) of patients with sickle cell disease. We propose to investigate the hypothesis the B-CAM/LU mediates both adhesion to laminin as well as cell-cell adhesion in sickle cell disease, thus playing an important role in the vaso- occlusive process leading to pain and organ damage. We further propose to see the results of these investigations and to explore therapeutically applicable methods for reduction of SS RBC adhesion to endothelium and subendothelial matrix laminin by developing reagents capable of interfering with B-CAM/LU binding to its ligands. First, we will use a variety of methods to identify the surface molecules of RBC and endothelial cells that serve as ligands in B-CAM/LU-mediated adhesion processes. Second, we will determine the process(es) involved in activation of B-CAM/LU adhesive function on S RBC and in nucleated cells expressing recombinant forms of B-CAM/LU. Both phosphorylation and protein- protein interactions may play a role in this process. Specifically, we will study possible interaction of B-CAM/LU with integrins (especially alpha4beta1) and with CD44 based on our preliminary data. We will also investigate the effect of serine/threonine phosphorylation within the cytoplasmic domain of B-CAM/LU, and whether this occurs to a different degree in SS versus normal RBC, and in oxygenated versus deoxygenated RBC. We will further determine if the putative SH3 binding motif of B-CAM/LU associates with either tyrosine kinases or other signaling molecules with SH3 motifs. Third, to develop specific inhibitors that block B-CAM/LU- dependent adhesion, we will use various adhesion assays to study the effects of anti-B-CAM/LU monoclonal antibodies, as well as soluble recombinant B-CAM/LU and inhibitory peptides identified through screening of peptide display phage libraries. Overall, these studies will further define how B-CAM/LU contributes to adhesion and vaso-occlusion and will identify avenues by which its interactions might be abrogated in a therapeutic setting.