Mechanisms of chemoattractant receptor regulation

描述（由申请人提供）：化学引诱剂及其受体是 我们免疫防御系统的关键组成部分，在吞噬细胞中起关键作用， 迁移和激活。“经典的”化学引诱剂包括甲酰化的 肽、补体片段C3a和C5a、血小板活化因子和 白三烯B4。它们结合具有显著序列同源性的受体 和结构特征，它们共同构成了G 蛋白偶联受体（GPCR）。该受体亚家族抑制每个 另一些则通过交叉脱敏发挥作用。在受体层次中 交叉脱敏，甲酰基肽受体（FPR）占主导地位 受体，即，FPR可使其他趋化因子受体交叉脱敏 比交叉脱敏FPR的程度更高。因此，我们也许能够 利用这些信息治疗慢性炎症 疾病通过了解受体激活的分子机制， 脱敏，我们可能会找到方法来钝化中性粒细胞对激活的反应， 信号. 我们以前的特点是突变FPR，表现出正常的配体 结合和G蛋白偶联，但在信号传导和 诱导趋化性。基于这一发现，我们提出， 除G蛋白外，其他蛋白质相互作用并调节FPR的功能。我们 将通过分析配体结合诱导的膜来验证这一假设 某些细胞质蛋白质的易位， 与其他GPCR相互作用。这将在CHO细胞中进行， 野生型FPR和各种突变型FPR。我们还将尝试识别 通过比较哪些蛋白质 与野生型共免疫沉淀，但不与突变型FPRs共免疫沉淀。与身份 以前未知的相互作用，我们将进行酵母双杂交 分析.我们将研究与FPR细胞质区域的相互作用， C5aR。正相互作用将通过哺乳动物双杂交证实 分析和免疫共沉淀。分子的功能，如果未知， 将通过在CHO中过表达全长或截短的蛋白质来检查 表达FPR的细胞。最后，我们将研究FPR的机制， 交叉脱敏C5aR。我们将检验我们的假设， C5aR的交叉脱敏需要FPR的脱敏。我们将 我们还验证了我们的假设，即C5aR与FPR的共内吞作用是一个重要的 C5aR的交叉脱敏和下调机制，而FPR的 对C5aR的优势可能部分是由于其对共内吞作用的抗性， 激活C5aR。这项工作应该提供新的信息， 化学引诱物受体介导的细胞活化的调节。

DESCRIPTION(provided by applicant): Chemoattractants and their receptors are key components of our immune defense system with a critical role in phagocyte migration and activation. The "classical" chemoattractants include formylated peptides, the complement fragments C3a and C5a, platelet-activating factor and leukotriene B4. They bind receptors that share significant sequence homology and structural features, and together they constitute a subfamily of G protein-coupled receptors (GPCR). This subfamily of receptors inhibit each others function through cross-desensitization. In the hierarchy of receptor cross-desensitization, the formyl peptide receptor (FPR) is the dominant receptor, i.e., FPR can cross-desensitize the other chemoattractant receptors to higher extent than they can cross-desensitize FPR. Thus, we may be able to take advantage of this information in treatment of chronic inflammatory diseases. By understanding the molecular mechanism of receptor activation and desensitization we may find ways to blunt the neutrophil response to activating signals. We have previously characterized a mutant FPR that exhibits normal ligand binding and G protein coupling, but shows functional defects in signaling and induction of chemotaxis. Based on this finding, we propose that cytoplasmic proteins, other than G proteins, interact and regulate the function of FPR. We will test this hypothesis by analyzing the ligand binding-induced membrane translocation of certain cytoplasmic proteins that have been previously shown to interact with other GPCRs. This will be carried out in CHO cells expressing wild-type FPR and various mutant FPRs. We will also attempt to identify additional interacting proteins by comparing which proteins are co-immunoprecipitated with wild-type, but not with mutant FPRs. To identity previously uncharacterized interactions, we will carry out yeast two-hybrid analysis. We will examine interactions with cytoplasmic regions of FPR and C5aR. The positive interactions will be confirmed by mammalian two-hybrid analysis and co-immunoprecipitation. The function of the molecules, if unknown, will be examined by overexpression of full length or truncated proteins in CHO cells expressing FPR. Finally, we will examine the mechanism by which FPR cross-desensitizes C5aR. We will test our hypothesis that homologous desensitization of FPR is required for cross-desensitization of C5aR. We will also test our hypothesis that co-endocytosis of C5aR with FPR is an important mechanism of cross-desensitization and down-regulation of C5aR, whereas FPR's dominance over C5aR may in part be due to its resistance to co-endocytosis upon activation of C5aR. This work should provide novel information regarding the regulation of chemoattractant receptor-mediated cell activation.