Cloning and Characterization of ATRAP, a Novel Protein That Interacts with the Angiotensin II Type 1 Receptor

Laurent Daviet,Jukka Y.A Lehtonen,Kouichi Tamura,Daniel P Griese,Masatsugu Horiuchi,Victor J Dzau

doi:10.1074/jbc.274.24.17058

Laurent Daviet, Jukka Y.A Lehtonen + Show 4 more

Open Access

https://doi.org/10.1074/jbc.274.24.17058

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Abstract

The carboxyl-terminal cytoplasmic domain of the angiotensin II type 1 (AT1) receptor has recently been shown to interact with several classes of cytoplasmic proteins that regulate different aspects of AT1 receptor physiology. Employing yeast two-hybrid screening of a mouse kidney cDNA library with the carboxyl-terminal cytoplasmic domain of the murine AT1a receptor as a bait, we have isolated a novel protein with a predicted molecular mass of 18 kDa, which we have named ATRAP (for AT1 receptor-associated protein). ATRAP interacts specifically with the carboxyl-terminal domain of the AT1a receptor but not with those of angiotensin II type 2 (AT2), m3 muscarinic acetylcholine, bradykinin B2, endothelin B, and beta2-adrenergic receptors. The mRNA of ATRAP was abundantly expressed in kidney, heart, and testis but was poorly expressed in lung, liver, spleen, and brain. The ATRAP-AT1a receptor association was confirmed by affinity chromatography, by specific co-immunoprecipitation of the two proteins, and by fluorescence microscopy, showing co-localization of these proteins in intact cells. Overexpression of ATRAP in COS-7 cells caused a marked inhibition of AT1a receptor-mediated activation of phospholipase C without affecting m3 receptor-mediated activation. In conclusion, we have isolated a novel protein that interacts specifically with the carboxyl-terminal cytoplasmic domain of the AT1a receptor and affects AT1a receptor signaling.

Highlights

G protein-coupled receptors (GPCRs)1 interact with different classes of intracellular proteins, including heterotrimeric G proteins, kinases, and arrestins [1,2,3]
As for many GPCRs, the carboxyl-terminal cytoplasmic domain of the angiotensin II type 1 (AT1) receptor presumably interacts with G protein-coupled receptor kinases and arrestins, causing functional desensitization of the receptor [18, 21]
These observations raise the possibility that the carboxylterminal cytoplasmic domain of the AT1 receptor interacts with additional cellular proteins that may play an important role in the efficacy and/or specificity of receptor-G protein coupling. We have investigated this possibility by searching for novel protein interactions with the carboxyl-terminal cytoplasmic domain of the murine AT1a receptor

Summary

Introduction

G protein-coupled receptors (GPCRs)1 interact with different classes of intracellular proteins, including heterotrimeric G proteins, kinases, and arrestins [1,2,3]. Co-immunoprecipitation—The NH2-terminal HA epitope-tagged ATRAP in pcDNA3 was transiently co-transfected with a FLAG-tagged murine AT1a receptor [24] in COS-7 cells according to the LipofectAMINE protocol (Life Technologies, Inc.). The yeast two-hybrid system was used to identify candidate proteins that interact with the carboxyl-terminal cytoplasmic tail of the mouse AT1a receptor.

Results

Conclusion