[Binding between alpha 1A-adrenergic receptor and segment of bone morphogenetic protein-1 in human embryonic cell 293].
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.
Xu
Qi
Q
Institute of Vascular Medicine, Peking University Third Hospital and Key Laboratory of Molecular Cardiology, Education Ministry, Beijing 100083.
Zhang
Tan
T
Han
Qi-De
QD
Zhang
You-Yi
YY
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D055395:Bone Morphogenetic Protein 1; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques
C547713:ADRA1A protein, human; D000316:Adrenergic alpha-Agonists; D019485:Bone Morphogenetic Proteins; D018340:Receptors, Adrenergic, alpha-1; D008666:Metalloendopeptidases; C525705:BMP1 protein, human; D055395:Bone Morphogenetic Protein 1