Novel Regulatory Effect of Angiotensin II Type 1 Receptor-Interacting Molecule on Vascular Smooth Muscle Cells

Abstract

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 ...

Atrap Deficiency Increases Arterial Blood Pressure and Plasma Volume

“…These data suggest that Atrap reduces proximal tubular AT1 surface expression, as shown before by in vitro studies. 14,16,18 Enhanced AngII binding to the proximal tubule of AtrapϪ/Ϫ mice would be expected to stimulate tubular salt and water reabsorption and promote acid excretion predominantly by stimulation of the Na/H exchanger NHE-3. 20,28,29 Thus, that we found lower urinary pH in AtrapϪ/Ϫ mice in comparison with wild-type mice would be in line with increased AngII-dependent NHE-3 activity in the proximal tubule of AtrapϪ/Ϫ mice.…”

“…15 Atrap catalyzes the internalization of the AT1 receptor in cultured vascular smooth muscle cells. 14,16 Also, Atrap inhibits AngII-mediated intracellular signaling in vitro. 17 Overall, the data suggest an inhibitory effect of Atrap on AT1 receptor function in vitro.…”

Analysis of genes causing hypertension and stroke in spontaneously hypertensive rats: Gene expression profiles in the brain

“…IPA of SHR-specific genes revealed that prostaglandin E receptor 4 (Ptger4) is one of the candidate genes responsible for causing hypertension in SHR (12,13), as well as albumin (Alb) and chymase 1 (Cma1), in the presence of angiotensinogen (Agt) (14)(15)(16). Similar analyses of SHRSPspecific genes revealed that angiotensin II receptor-associated gene (Agtrap) interacts with FBJ osteosarcoma oncogene (Fos), and with angiotensin II receptor type-1B (Agtr1b) (17)(18)(19). These interactions play pivotal roles among SHRSP-specific genes, and since Agtrap and Agtr1b not only participate in the 'uptake of norepinephrine' and 'blood pressure', but also in the 'behavior' of 6-week-old SHRSP, the data presented in the present study reveal a close association between hypertension and ADHD.…”

Effect of Olmesartan on Tissue Expression Balance Between Angiotensin II Receptor and Its Inhibitory Binding Molecule

“…19 -25 The results of our previous in vitro studies also showed that ATRAP suppresses Ang II-induced hypertrophic and proliferative responses by promoting a constitutive internalization of AT1R and decreasing the p38 MAPK activity in cardiovascular cells, thereby suggesting ATRAP to be an endogenous inhibitor of AT1R signaling. 6,12 In this study, it is revealed that the incubation of cardiomyocytes with Ang II as well as the chronic infusion of Ang II into Sprague-Dawley rats significantly decreased the ratio of ATRAP to AT1R through a suppression of ATRAP protein expression concomitantly with the hypertrophic responses in vitro and in vivo. We observed that olmesartan abolished Ang II-mediated decreases in the ATRAP to AT1R ratio through an upregulation of ATRAP protein expression in mice cardiomyocytes and hearts of Ang II-infused mice (Wakui and Tamura, unpublished observation).…”

“…5,6,12 The anti-AT1R antibody (sc-1173) was purchased from Santa Cruz Biotechnology, Inc. 5 Western blot analysis was performed as described previously. 5,6 Briefly, the cell extracts (20 g per lane) or rat tissue extracts (10 g per lane for liver and kidney and 20 g per lane for heart) were used for electrophoresis.…”