FACTORS INVOLVED IN B-CAM/LU MEDIATED ADHESION

B-CAM/LU是一种粘附分子，其在上皮癌和镰状细胞病患者的红细胞（RBC）上的表达增加。我们建议研究B-CAM/LU介导镰状细胞病中与层粘连蛋白的粘附以及细胞-细胞粘附的假设，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调查的结果，并探索治疗上适用的方法，通过开发能够干扰B-CAM/LU与其配体结合的试剂，减少SS RBC与内皮和内皮下基质层粘连蛋白的粘附。首先，我们将使用各种方法来确定红细胞和内皮细胞的表面分子，作为B-CAM/LU介导的粘附过程中的配体。其次，我们将确定参与激活S RBC上和表达重组形式B-CAM/LU的有核细胞中B-CAM/LU粘附功能的过程。磷酸化和蛋白质-蛋白质相互作用可能在这一过程中发挥作用。具体来说，我们将研究可能的相互作用B-CAM/LU与整合素（特别是α 4 β 1）和CD 44的基础上，我们的初步数据。我们还将研究B-CAM/LU胞质结构域内丝氨酸/苏氨酸磷酸化的影响，以及这是否在SS与正常RBC中以及在氧合与脱氧RBC中发生不同程度。我们将进一步确定B-CAM/LU的假定SH 3结合基序是否与酪氨酸激酶或其他具有SH 3基序的信号分子相关。第三，为了开发阻断B-CAM/LU依赖性粘附的特异性抑制剂，我们将使用各种粘附测定来研究抗B-CAM/LU单克隆抗体的作用，以及可溶性重组B-CAM/LU和通过肽展示噬菌体文库筛选鉴定的抑制肽。总之，这些研究将进一步确定B-CAM/LU如何促进粘连和血管闭塞，并将确定在治疗环境中消除其相互作用的途径。

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.