Lafuente Esther, Boussiotis Vassiliki A
Transplantation Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Methods Enzymol. 2006;407:345-58. doi: 10.1016/S0076-6879(05)07029-1.
The small GTPase Rap1 has been involved in different cellular processes. Rap1 is known to increase cell adhesion by means of integrin activation, to induce cell spreading, and to regulate adherent junctions at cell-cell contacts. How Rap1 mediates these cell responses is poorly known, but currently developing evidence points to the involvement of different effector pathways. Recently, we described RIAM, a Rap1 interacting adaptor protein that regulates integrin activation and hence cell adhesion. RIAM is required for Rap1-induced adhesion and seems to control Rap1 localization at the plasma membrane, where Rap1 regulates integrin activation. In this chapter, we focus in the role of RIAM in regulating Rap1-mediated cell adhesion. We describe the method for studying the Rap1-RIAM interaction using in vitro and in vivo approaches such as yeast two hybrids, pull-down assays. and coimmunoprecipitation. The role of Rap1 and RIAM in integrin-mediated adhesion is studied by cell adhesion assays to immobilized integrin substrates and by changes in integrin activation as determined by activation epitope exposure. Finally, we describe an approach to determine the role of RIAM in regulating intracellular localization of active Rap1.
小GTP酶Rap1参与了不同的细胞过程。已知Rap1通过整合素激活来增强细胞黏附，诱导细胞铺展，并调节细胞间接触处的黏附连接。Rap1如何介导这些细胞反应尚不清楚，但目前不断有证据表明不同的效应器途径参与其中。最近，我们描述了RIAM，一种与Rap1相互作用的衔接蛋白，它调节整合素激活，进而调节细胞黏附。RIAM是Rap1诱导黏附所必需的，似乎能控制Rap1在质膜上的定位，而Rap1在质膜上调节整合素激活。在本章中，我们重点关注RIAM在调节Rap1介导的细胞黏附中的作用。我们描述了使用体外和体内方法（如酵母双杂交、下拉试验和共免疫沉淀）研究Rap1-RIAM相互作用的方法。通过对固定化整合素底物的细胞黏附试验以及通过激活表位暴露所确定的整合素激活变化，研究了Rap1和RIAM在整合素介导的黏附中的作用。最后，我们描述了一种确定RIAM在调节活性Rap1细胞内定位中作用的方法。
