Biener Eva, Martin Cyril, Daniel Nathalie, Frank Stuart J, Centonze Victoria E, Herman Brian, Djiane Jean, Gertler Arieh
The Institute of Biochemistry, Food Science, and Nutrition, Faculty of Agricultural, and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel.
Endocrinology. 2003 Aug;144(8):3532-40. doi: 10.1210/en.2003-0096.
HEK-293T cells transiently transfected with ovine (o) GH receptor (GHR) and prolactin receptor (PRLR) constructs respectively tagged downstream with cyan or yellow fluorescent proteins were used to study ovine placental lactogen (oPL)-stimulated heterodimerization by fluorescence resonance energy transfer (FRET) microscopy. The oPL-stimulated transient heterodimerization of GHR and PRLR had a peak occurring 2.5-3 min after oPL application, whereas oGH or oPRL had no effect at all. The results indicate none or only little dimerization occurring before the hormonal stimulation. The effect of heterodimerization was studied by comparing activation of Janus kinase 2, signal transducer and activator of transcription (STAT)1, STAT3, STAT5, and MAPK in Chinese hamster ovary cells stably transfected with chimeric genes encoding receptors consisting of cytosolic and transmembrane parts of oGHR and oPRLR, extracellular domains of human granulocyte and macrophage colony-stimulating factor (hGM-CSF) receptor alpha or beta, and cells transfected with the two forms (alpha or beta) of PRLR and GHR. Functionality of those proteins was verified by hGM-CSF-induced phosphorylation of both intracellular PRLR and GHR domains and hGM-CSF-induced heterodimerization was documented by chimeric receptor coimmunoprecipitation. Homodimerization or heterodimerization of PRLRs and GHRs had no differential effect on activation of STAT5 and MAPK. However, heterodimerization resulted in a prolonged phosphorylation of STAT1 and in particular STAT3, suggesting that the heterodimerization of alpha-oGHR and beta-oPRLR is able to transduce a signal, which is distinct from that occurring on homodimeric associations.
将分别用青色或黄色荧光蛋白标记下游的绵羊（o）生长激素受体（GHR）和催乳素受体（PRLR）构建体瞬时转染的HEK-293T细胞，用于通过荧光共振能量转移（FRET）显微镜研究绵羊胎盘催乳素（oPL）刺激的异二聚化。oPL刺激的GHR和PRLR瞬时异二聚化在施加oPL后2.5 - 3分钟出现峰值，而oGH或oPRL则完全没有作用。结果表明在激素刺激之前没有或仅有少量二聚化发生。通过比较在中国仓鼠卵巢细胞中稳定转染嵌合基因的Janus激酶2、信号转导和转录激活因子（STAT）1、STAT3、STAT5和丝裂原活化蛋白激酶（MAPK）的激活情况，研究异二聚化的作用，这些嵌合基因编码的受体由oGHR和oPRLR的胞质和跨膜部分、人粒细胞和巨噬细胞集落刺激因子（hGM-CSF）受体α或β的细胞外结构域组成，以及用PRLR和GHR的两种形式（α或β）转染的细胞。通过hGM-CSF诱导的细胞内PRLR和GHR结构域的磷酸化验证了这些蛋白质的功能，并且通过嵌合受体共免疫沉淀记录了hGM-CSF诱导的异二聚化。PRLR和GHR的同二聚化或异二聚化对STAT5和MAPK的激活没有差异影响。然而，异二聚化导致STAT1特别是STAT3的磷酸化延长，这表明α-oGHR和β-oPRLR的异二聚化能够转导一种与同二聚体结合时不同的信号。
