Abstract-We cloned a novel molecule interacting with angiotensin II type 1 receptor, which we named ATRAP (for angiotensin II type 1 receptor-associated protein). Previous in vitro studies showed that ATRAP significantly promotes constitutive internalization of the angiotensin II type 1 receptor and further attenuates angiotensin II-mediated hypertrophic responses in cardiomyocytes. The present study was designed to investigate the putative functional role of ATRAP in cardiac hypertrophy by angiotensin II infusion in vivo. We first examined the effect of angiotensin II infusion on endogenous ATRAP expression in the heart of C57BL/6J wild-type mice. The angiotensin II treatment promoted cardiac hypertrophy, concomitant with a significant decrease in cardiac ATRAP expression, but without significant change in cardiac angiotensin II type 1 receptor expression. We hypothesized that a downregulation of the cardiac ATRAP to angiotensin II type 1 receptor ratio is involved in the pathogenesis of cardiac hypertrophy. To examine this hypothesis, we next generated transgenic mice expressing ATRAP specifically in cardiomyocytes under control of the ␣-myosin heavy chain promoter. In cardiac-specific ATRAP transgenic mice, the development of cardiac hypertrophy, activation of p38 mitogen-activated protein kinase, and expression of hypertrophy-related genes in the context of angiotensin II treatment were completely suppressed, in spite of there being no significant difference in blood pressure on radiotelemetry between the transgenic mice and littermate control mice. These results demonstrate that cardiomyocyte-specific overexpression of ATRAP in vivo abolishes the cardiac hypertrophy provoked by chronic angiotensin II infusion, thereby suggesting ATRAP to be a novel therapeutic target in cardiac hypertrophy. (Hypertension. 2010;55:1157-1164.)Key Words: basic science Ⅲ receptors Ⅲ gene expression/regulation Ⅲ hypertrophy/remodeling Ⅲ angiotensin receptors E vidence suggests that the activation of angiotensin II (Ang II) type 1 receptor (AT 1 R) through the tissue renin-angiotensin system may play an important role in the development of cardiac hypertrophy. The carboxyl-terminal portion of AT 1 R is involved in the control of AT 1 R internalization independent of G protein coupling, and it plays an important role in linking receptor-mediated signal transduction to the specific biological response to Ang II. 1,2 We previously cloned a novel AT 1 R-associated protein (ATRAP) that specifically interacts with the carboxylterminal domain of AT 1 R. [3][4][5][6] We showed that ATRAP is broadly expressed in many tissues, as is AT 1 R, and suppresses Ang II-mediated pathological responses in cardiomyocytes and vascular smooth muscle cells by promoting the constitutive internalization of AT 1 R. 7-9 However, the function of ATRAP in cardiac hypertrophy in vivo still remains to be demonstrated. Thus, the present study was carried out to investigate whether there is a role for ATRAP in the cardiac hypertrophy induced by chronic Ang II ...

We have previously shown that angiotensin II type 1 receptor-associated protein (ATRAP/Agtrap) interacts with the angiotensin II type 1 receptor and promotes constitutive internalization of the receptor so as to inhibit the pathological activation of its downstream signaling but preserve baseline physiological signaling activity. The present study was designed to investigate the role of renal ATRAP in angiotensin II–dependent hypertension. We generated transgenic mice dominantly expressing ATRAP in the renal tubules, including renal distal tubules. The renal ATRAP transgenic mice exhibited no significant change in blood pressure at baseline on normal salt diet. However, in the renal ATRAP transgenic mice compared with wild-type mice, the following took place: (1) the development of high blood pressure in response to angiotensin II infusion was significantly suppressed based on radiotelemetry, (2) the extent of daily positive sodium balance was significantly reduced during angiotensin II infusion in metabolic cage analysis, and (3) the renal Na+-Cl− cotransporter activation and α-subunit of the epithelial sodium channel induction by angiotensin II infusion were inhibited. Furthermore, adenoviral overexpression of ATRAP suppressed the angiotensin II–mediated increase in the expression of α-subunit of the epithelial sodium channel in mouse distal convoluted tubule cells. These results indicate that renal tubule–dominant ATRAP activation provokes no evident effects on blood pressure at baseline but exerts an inhibitory effect on the pathological elevation of blood pressure in response to angiotensin II stimulation, thereby suggesting that ATRAP is a potential target of interest in blood pressure modulation under pathological conditions.

Abstract-We have recently cloned a novel molecule that interacts with the angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP).In this study, we tested the hypothesis that ATRAP modulates angiotensin II-induced responses in vascular smooth muscle cells. The results of immunoprecipitation and bioluminescence resonance energy transfer assay demonstrated a direct interaction between ATRAP and AT1R at baseline and showed that angiotensin II enhanced the interaction of these proteins Ͼ2-fold. The results of immunofluorescence analysis also demonstrated that Ͼ65% of ATRAP constitutively colocalized with an endosome marker. Although only 36% of ATRAP colocalized with AT1R at baseline, angiotensin II enhanced the colocalization of these molecules and made 92% of ATRAP colocalize with AT1R on a quantitative fluorescence analysis. Overexpression of ATRAP by adenoviral transfer decreased the cell surface AT1R number from 4.33 to 2.13 fmol/10 6 cells at baseline and from 3.04 to 1.26 fmol/10 6 cells even after removal of angiotensin II. ATRAP also suppressed angiotensin II-mediated increases in c-fos gene transcription and transforming growth factor-␤ production. Furthermore, this suppression was accompanied by inhibition of angiotensin II-induced activation of 5-bromodeoxyuridine incorporation. Finally, ATRAP knockdown by small-interference RNA activated angiotensin II-induced c-fos gene expression, which was effectively inhibited by valsartan, an AT1R-specific antagonist. These results indicate that ATRAP promotes internalization of AT1R and attenuates the angiotensin II-mediated c-fos-transforming growth factor-␤ pathway and proliferative response in vascular smooth muscle cells, suggesting a novel strategy to inhibit vascular fibrosis and remodeling through a novel and specific blockade of AT1R signaling. (AT1R) is a member of the G protein-coupled receptor superfamily and activates G proteins through the third intracellular loop and the intracellular carboxyl-terminal (C-terminal) tail of the receptor. 1,2 The C-terminal cytoplasmic end of AT1R is involved in the control of AT1R internalization independent of G protein coupling, and it plays an important role in linking receptormediated signal transduction to the specific biological response to angiotensin II (Ang II), such as cardiovascular fibrosis and remodeling. 3,4 Using a yeast 2-hybrid screening system, we recently cloned a novel AT1R-associated protein (ATRAP) that specifically interacts with the C-terminal cytoplasmic domain of AT1R. [5][6][7] We showed that ATRAP is expressed in a variety of tissues and suppresses Ang IImediated hypertrophic responses in cardiac myocytes. 8,9 ARAP1 is another protein that was found recently to interact with the C-terminal domain of AT1R. 10 Characterization of ARAP1 has revealed that ARAP1 binds and promotes recycling of AT1R to the plasma membrane, indicating its role in the receptor-recycling pathway. In this study, we examined the function of ATRAP in Ang II-induced fibrotic and proliferative responses of rat ...