Yeast two-hybrid screening for proteins that interact with alpha1-adrenergic receptors.

Abstract:

AIM: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). CONCLUSION: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.

journal_name

Acta Pharmacol Sin

authors

Zhang T,Xu Q,Chen FR,Han QD,Zhang YY

subject

Has Abstract

pub_date

2004-11-01 00:00:00

pages

1471-8

issue

11

eissn

1671-4083

issn

1745-7254

journal_volume

25

pub_type

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