Novel regulatory effect of angiotensin II type 1 receptor-interacting molecule on vascular smooth muscle cells.
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 >2-fold. The results of immunofluorescence analysis also demonstrated that >65% 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-beta 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-beta 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.
Azuma
Koichi
K
Department of Cardiorenal Medicine, Yokohama City University School of Medicine, Yokohama, Japan.
Tamura
Kouichi
K
Shigenaga
Atsu-ichiro
A
Wakui
Hiromichi
H
Masuda
Shin-ichiro
S
Tsurumi-Ikeya
Yuko
Y
Tanaka
Yutaka
Y
Sakai
Masashi
M
Matsuda
Miyuki
M
Hashimoto
Tatsuo
T
Ishigami
Tomoaki
T
Lopez-Ilasaca
Marco
M
Umemura
Satoshi
S
D000804:Angiotensin II / Q000494:pharmacology; D047228:Angiotensin II Type 1 Receptor Blockers / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D031541:Fluorescence Resonance Energy Transfer; D018014:Gene Transfer Techniques; D047468:Immunoprecipitation; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D044140:Receptor, Angiotensin, Type 1 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011945:Receptors, Angiotensin / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013777:Tetrazoles / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014633:Valine / Q000031:analogs & derivatives / Q000494:pharmacology; D000068756:Valsartan
C524087:Agtrap protein, rat; D047228:Angiotensin II Type 1 Receptor Blockers; D016760:Proto-Oncogene Proteins c-fos; D034741:RNA, Small Interfering; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; D013777:Tetrazoles; D000804:Angiotensin II; D000068756:Valsartan; D014633:Valine