Binding between alpha 1A-adrenergic receptor and segment of bone morphogenetic protein-1 in human embryonic cell 293.
- Author:
Qi XU
1
;
Tan ZHANG
;
Qi-De HAN
;
You-Yi ZHANG
Author Information
1. Institute of Vascular Medicine, Peking University Third Hospital and Key Laboratory of Molecular Cardiology, Education Ministry, Beijing 100083.
- Publication Type:Journal Article
- MeSH:
Adrenergic alpha-Agonists;
pharmacology;
Bone Morphogenetic Protein 1;
Bone Morphogenetic Proteins;
genetics;
metabolism;
Cells, Cultured;
Embryo, Mammalian;
Enzyme-Linked Immunosorbent Assay;
Humans;
Kidney;
cytology;
metabolism;
Metalloendopeptidases;
genetics;
metabolism;
Protein Binding;
Receptors, Adrenergic, alpha-1;
genetics;
metabolism;
Transfection;
Two-Hybrid System Techniques
- From:
Acta Physiologica Sinica
2003;55(6):692-698
- CountryChina
- Language:Chinese
-
Abstract:
Using matchmaker yeast two-hybrid system, it has been demonstrated that there exists an interaction between the cellular C terminal of alpha(1A)-adrenergic receptor (alpha(1A)-AR) and a segment of bone morphogenetic 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-alpha(1A) consisted of the full-length cDNA of human alpha(1A)-AR. They were transfected to HEK293 cells and examined by Western blot. alpha(1A)-AR and the segment of BMP-1 could be detected in the lysis of transfected cells. Then binding between alpha(1A)-AR and the segment of BMP-1 in HEK293 cell was determined by enzyme-linked immunosorbent 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 alpha(1A)-AR, then the cell lysis, anti-HA rabbit polyclonal antibody and HRP conjugated anti-rabbit antibody were added in turn. The OD(490) values among the control group, PDT-alpha(1A) transfection group and PCP3HA transfection group, exhibited no significant difference (0.034+/-0.027, 0.042+/-0.019, 0.030+/-0.0096), but the OD(490) values of PDT-alpha(1A) 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 alpha(1A)-AR or/and segment of BMP-1 were lysed and incubated with anti-FLAG M2 antibody, then the immunoprecipitation pellet was immunoblotted 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-alpha(1A) and PCP3HA co-transfected group. In conclusion, the results indicate that alpha(1A)-AR and the segment of BMP-1 are present in the same complex in HEK293 cells.
