Study on molecular mechanisms of integrin-regulated adhesion in immune responses

我们已经报道，小GTPase Rap1是一种细胞内信号分子，可以增加整合素的粘附性，这种粘附性是由趋化因子或抗原刺激触发的。趋化因子激活RAP1有力地刺激淋巴细胞的运动。我们还证明了在生理剪切流下，趋化因子激活Rap1是淋巴细胞通过内皮单分子层迁移所必需的。此外，Rap1激活诱导细胞极化，伴随着前缘和尾足的发育以及趋化因子受体CXCR4和CD44分别重新定位到前缘和尾足。为了阐明这些过程中的分子机制，我们鉴定并表征了一种新的Rap1效应器RAPL。RAPL是一种与Rap1-GTP结合的30kD蛋白，通过酵母双杂交筛选得到一个活性的Rap1突变体。RAPL主要表达于脾、淋巴结和胸腺，尤其是T和B细胞。RAPL过表达可刺激LFA-1介导的ICAM-1黏附，而N端缺失突变体为显性负性突变体，可抑制TCR和趋化因子诱导的ICAM-1黏附。RAPL增强LFA-1的配体结合亲和力和淋巴细胞极化。在趋化因子的刺激下，RAPL与LFA-1形成复合体，并根据Rap1的激活而移位到前沿。综上所述，这些结果表明，当RAPL与Rap1-GTP结合时，它诱导细胞极化并移位LFA-1，通过调节LFA-1的亲和力和聚集性而导致粘附性增加。

We have reported that the small GTPase Rap1 is a intracellular signaling molecule that increase integrin adhesiveness that is triggered by stimulation with chemokines or antigens. Rap1 activation by chemokines robustly stimulates lymphocyte motility. We have also demonstrated that Rap1 activation by chemokines is required in lymphocyte transmigration through endothelial monolayers under physiological shear flow. Furthermore, Rap1 activation induces cell polarization, which is accompanied with development of the leading edge and uropod and relocalization of chemokine receptor CXCR4 and CD44 to the leading edge and uropod, respectively. In order to clarigy the molecular mechanism in these processes, we have identified and characterized a novel Rap1 effector RAPL. RAPL is a Rap1-GTP binding 30 kD protein isolated by yeast two-hybrid screening with an active Rap1 mutant. RAPL is preferentially expressed in spleen, lymph nodes, and thymus, in particular T and B cells. RAPL overexpression stimulated LFA-1-mediated adhesion to ICAM-1, whereas a N-terminal deletion mutant acted as a dominant negative mutant and inhibited adhesion to ICAM-1 triggered by TCR and chemokines. RAPL augment ligand-binding affinity of LFA-1 and lymphocyte polarization. Upon stimulation with chemokines, RAPL forms a complex with LFA-1 and translocates to the leading edge, depending on Rap1 activation. Taken together, these results indicate that when RAPL binds to Rap1-GTP, it induce cell polarization and translocates LFA-1, resulting in an increase of adhesiveness through modulation of affinity and clustering of LFA-1.