Yeast two-hybrid screening for proteins that interact with alpha1-adrenergic receptors.
OBJECTIVE
To find novel proteins that may bind to alpha1A-adrenergic receptor (alpha1A-AR) and investigate their interactions with the other two alpha1-AR subtypes (alpha1B-AR and alpha1D-AR) with an expectation to provide new leads for the function study of the receptors.


METHODS
Yeast two-hybrid assay was performed to screen a human brain cDNA library using the C terminus of alpha1A-AR (alpha1A-AR-CT) as bait. X-Gal assay and o-nitrophenyl-beta-D-galactopyranoside (ONPG) assay were subsequently conducted to further qualitatively or quantitatively confirm the interactions between receptors and the three identified proteins.


RESULTS
(1) Selection medium screening identified segments of bone morphogenetic protein-1 (BMP-1), active Bcr-related protein (Abr), and filamin-C as binding partners of alpha1A-AR-CT in yeast cells respectively. Besides, protein segments of BMP-1 and Abr could only specifically interact with alpha1A-AR-CT while filamin-C segment interacted with all three alpha1-AR subtypes. (2) In X-Gal assay, the co-transformants of alpha1A-AR-CT and BMP-1 segments turned strong blue at about 30 min while other positive transformants only developed weak blue at about 5-6 h. (3) In ONPG assay, interaction (shown in beta-galactosidase activity) between alpha1A-AR-CT and BMP-1 segments was about 30 times stronger than that of control (P<0.01), while other positive interactions were only about 2-5 times as strong as those of controls (P<0.05).


CONCLUSIONS
In yeast cells BMP-1, Abr and/or filamin-C could interact with three alpha1-AR subtypes, among which, interaction between BMP-1 and alpha1A-AR was the strongest while other interactions between proteins and receptors were relatively weak.
Zhang
Tan
T
Institute of Vascular Medicine, Peking University Third Hospital and Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing, China.
Xu
Qi
Q
Chen
Feng-rong
FR
Han
Qi-de
QD
Zhang
You-yi
YY
D055395:Bone Morphogenetic Protein 1; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism*; D003285:Contractile Proteins / Q000235:genetics / Q000378:metabolism; D064448:Filamins; D020690:GTPase-Activating Proteins; D015723:Gene Library; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic; D015003:Yeasts / Q000235:genetics* / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
C082659:ABR protein, human; C547713:ADRA1A protein, human; C547716:ADRA1B protein, human; C547719:ADRA1D protein, human; D019485:Bone Morphogenetic Proteins; D003285:Contractile Proteins; D064448:Filamins; D020690:GTPase-Activating Proteins; D008840:Microfilament Proteins; D011506:Proteins; D018340:Receptors, Adrenergic, alpha-1; D001616:beta-Galactosidase; D008666:Metalloendopeptidases; C525705:BMP1 protein, human; D055395:Bone Morphogenetic Protein 1