摘要
用酵母双杂交方法发现骨形成蛋白-1(bone morphogenetic protein-1,BMP-1)的片段可以与α1A-肾上腺素受体(adrenergic receptor,AR)的细胞内游离C末端结合。进一步探讨了二者在哺乳动物表达系统人胚肾293细胞(human embryonic cell 293,HEK293)中的相互作用。采用PCR方法构建含BMP-1片段eDNA的真核表达质粒PCP3HA,将其与含全长人α1A-AR eDNA的质粒PDT-α1A分别或共同转染HEK293细胞,用免疫印迹法检测到α1A-AR和BMP-1在HEK293细胞有相应的蛋白表达。用酶联免疫吸附实验对免疫印迹鉴定过的细胞裂解液进行检测,观察到空白对照组,单独转染PDTα1A-和单独转染PCP3HA的细胞,OD490值分别为0.034±0.027、0.042±0.019、0.030±0.0096,三者之间无显著性差异。共转染PDT-α1A和PCP3HA的细胞,OD490值为0.57±0.12,较其它三组具有显著性差异(均P<0.001)。免疫共沉淀结果显示,单独转染PDT-α1A或PCP3HA的细胞,免疫沉淀产物中均不能检测到BMP-1片段,只有共转染PDT-α1A和PCP3HA的细胞,在其免疫沉淀产物中能检测到BMP-1片段。ELISA和免疫沉淀结果均表明α1A-AR与BMP-1的片段在HEK293细胞中存在蛋白水平的相互作用。
Using matchmaker yeast two-hybrid system, it has been demonstrated that there exists an interaction between the cellular C terminal of α1A-adrenergic receptor (α1A-AR) and a segment of bone mor-phogenetic protein-1 ( BMP-1 ). In the present study binding between the two proteins was further determined in human embryonic cell 293 ( HEK293 ) , a mammalian expression system. Mammalian expression vector PCP3HA was constructed by PCR and consisted of segments of BMP-1 cDNA, and vector PDT-α1A consisted of the full-length cDNA of human α1A-AR. They were transfected to HEK293 cells and examined by Western blot. α1A-AR and the segment of BMP-1 could be detected in the lysis of transfected cells. Then binding betweenα1A-AR and the segment of BMP-1 in HEK293 cell was determined by enzyme-linked im-munosorbent assays ( ELISA) and co-immunoprecipitation. In ELISA experiment, the ELISA microwell plate was first coated with anti-FLAG M2 antibody, which recognizes the FLAG-tagged α1A-AR, then the cell lysis, anti-HA rabbit polyclonal antibody and HRP conjugated anti-rabbit antibody were added in turn. The OD490 values among the control group, PDT-α1A transfection group and PCP3HA transfection group, ex- hibited no significant difference (0. 034 ±0. 027 , 0. 042 ±0. 019, 0. 030 ±0. 0096) , but the OD490 values of PDT-α1A and PCP3HA co-transfection group (0. 57±0. 12) were significantly higher than those of the other groups (P<0. 001, respectively). In co-immunoprecipitation experiments, HEK293 cells expressing α1A-AR or/and segment of BMP-1 were lysed and incubated with anti-FLAG M2 antibody, then the immu-noprecipitation pellet was Jmmunoblotted with either the HRP conjugated anti-FLAG antibody or the anti-HA antibody, which recognizes the HA-tagged segment of BMP-1. Segment of BMP-1 was present in the pellet immunoprecipitation of PDT-α1A and PCP3HA co-transfected group. In conclusion, the results indicate that α1A-AR and the segment of BMP-1 are present in the same complex in HEK293 cells.
出处
《生理学报》
CAS
CSCD
北大核心
2003年第6期692-698,共7页
Acta Physiologica Sinica
基金
This work was supported by the National Basic Research Priorities Programme of China (No. G2000056906)
the National Natural Science Foundation of China (No. 30270540
30171083).