Cloning and characterization of ATRAP, a novel protein that interacts with the angiotensin II type 1 receptor.
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.
Daviet
L
L
Department of Medicine, Harvard Medical School, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Lehtonen
J Y
JY
Tamura
K
K
Griese
D P
DP
Horiuchi
M
M
Dzau
V J
VJ
D015059:1-Sarcosine-8-Isoleucine Angiotensin II / Q000031:analogs & derivatives / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014018:Tissue Distribution; D010738:Type C Phospholipases / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C119712:Agtrap protein, mouse; D002352:Carrier Proteins; D018076:DNA, Complementary; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D011994:Recombinant Proteins; C099368:angiotensin II, iodo-(Sar(1)-Ile(8))-; D000804:Angiotensin II; D015059:1-Sarcosine-8-Isoleucine Angiotensin II; D010738:Type C Phospholipases