Ames R S, Nuthulaganti P, Kumar C
Department of Molecular Immunology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406-0939, USA.
FEBS Lett. 1996 Oct 21;395(2-3):157-9. doi: 10.1016/0014-5793(96)01018-6.
The Xenopus laevis oocyte has been widely utilized for cloning and functional expression of G-protein coupled receptors (GPCR). This system was used for the functional expression and characterization of the recently identified human C3a receptor. Complementary RNA from the human C3a receptor was transcribed in vitro and microinjected into Xenopus oocytes for functional characterization. A positive response to a synthetic C3a peptide agonist and to C3a, but not to platelet activating factor or fMetLeuPhe was detected. In addition, a response of approximately one third the amplitude obtained with C3a was obtained with rC5a. Conversely, oocytes co-injected with the C5a receptor and total RNA isolated from U937 cells responded to C5a as well as to C3a and the C3a synthetic peptide. A functional response with the anaphylatoxin C3a receptor in oocytes was dependent on co-injection of a pertussis toxin sensitive complementary human factor which could be supplied by co-injection of total RNA isolated from U937 cells. Oocytes expressing the anaphylatoxin C3a and C5a receptors responded to both agonists, in each case the response to the cognate ligand was substantially more robust than the response elicited by the other anaphylatoxin.
非洲爪蟾卵母细胞已被广泛用于G蛋白偶联受体(GPCR)的克隆和功能表达。该系统用于最近鉴定出的人C3a受体的功能表达和特性研究。人C3a受体的互补RNA在体外转录,并显微注射到非洲爪蟾卵母细胞中进行功能特性分析。检测到对合成C3a肽激动剂和C3a有阳性反应,但对血小板活化因子或fMetLeuPhe没有反应。此外,用rC5a获得的反应幅度约为用C3a获得的三分之一。相反,与C5a受体共注射以及从U937细胞分离的总RNA的卵母细胞对C5a以及C3a和C3a合成肽有反应。卵母细胞中过敏毒素C3a受体的功能反应取决于共注射一种对百日咳毒素敏感的互补人因子,该因子可通过共注射从U937细胞分离的总RNA来提供。表达过敏毒素C3a和C5a受体的卵母细胞对两种激动剂都有反应,在每种情况下,对同源配体的反应比对另一种过敏毒素引发的反应要强得多。