ANALYSES OF THE SIGNAL TRANSDUCTION OF NOVEL ANTI-ARTHRITIC FACTOR, FRP, AND ITS EFFECTS ON IMMUNE SYSTEM

1.FRP受体A7的分子相互作用分析：我们的Biacore研究表明，FRP实际上与A7和CD14结合，也与激活素A、卵泡抑素、转化生长因子-β1结合。KD值分别为：FRP和A7为4.20×10^-6；-4；M，FRP和CD14为1.72×10；-4；-6，FRP和激活素A为1.43×10；-6，FRP和卵泡抑素为1.88×10；-4，FRP和转化生长因子-β1为1.28×10；-8；M。其他结合对的K_D值无法确定。FRP的特点是除转化生长因子-β外，对伴侣分子具有极高的速度和相对较低的亲和力结合活性。激活素A和CD14不与A7结合，而卵泡抑素与A7结合较慢，并干扰FRP与A7的结合。有趣的是，转化生长因子-β1也与A7具有较高的亲和力。然而，从其特征的矩形感觉图来看，结合的转化生长因子-β1似乎被更多的FRP所取代。FRP对转化生长因子-β1与转化生长因子-β1受体的结合有轻微的干扰作用，竞争作用很小，因此，似乎对转化生长因子-β信号转导几乎没有抑制作用。综上所述，FRP似乎与转化生长因子-β家族蛋白相互作用，从而在包括免疫系统在内的组织形成中发挥重要作用。FRP信号系统中第二信使的克隆：所有的双杂交克隆细胞SA240、SA263、SB224、SC21、SC23都被证明缺乏猎物载体，可能是因为它们的产物蛋白对宿主细胞有毒性。这促使我们考虑使用另一种双杂交系统进行克隆，不是使用酵母，而是使用细菌，因为细菌的物种与哺乳动物的距离更远。

1. Molecular interaction analysis of FRP receptor, A7: Our BIACORE studies disclosed that FRP actually bound to A7 and CD14, and also to activin A, follistatin, TGF-beta1. Their K_D values were calculated as follows: 4.20×10^<-6>M for FRP and A7, 1.72×10^<-4>M for FRP and CD14, 1.43×10^<-6>M for FRP and activin A, 1.88×10^<-4>M for FRP and follistatin, 1.28×10^<-8>M for FRP and TGF-beta1. K_D values for other binding pairs could not be decided. FRP was characterized by having extremely high speed and comparatively low affinity binding activity for partner molecules except for TGF-beta. Activin A and CD14 didn't bind to A7, however, follistatin bound weekly and quite slowly to A7, and they all interfered with FRP binding to A7. Interestingly, TGF-beta1 also bound to A7 with relatively-high affinity. However, bound TGF-beta1 seemed to be substituted by a larger amount of FRP, judging from its characteristic rectangular sensorgram. FRP trivially interfered TGF-beta1 binding to TGF-beta1sRII with a little competition, therefore, it seemed that FRP could hardly impede TGF-beta signaling. Taken together, FRP seems to interact with TGF-beta family proteins, and thereby play an important part in the tissue formation including the immune system.2. Cloning of second messengers in FRP signaling system: All of the resultant two-hybrid clone cells, SA240, SA263, SB224, SC21, SC23 proved to lack prey vectors, probably because their product proteins could be toxic to host cells. This brought us to consider cloning by another two-hybrid system not with yeast but with bacteria, whose species is more distant from the mammals.