Identification of calcium-modulating cyclophilin ligand (CAML) as transducer of angiotensin II-mediated nuclear factor of activated T cells (NFAT) activation.
Angiotensin II (Ang II) plays a central role in cardiovascular physiology and disease. Ang II type I receptor (AT1) is thought to mediate most actions of Ang II. A novel AT1 receptor intracellular partner called AT1 receptor-associated protein (ATRAP) was identified, but its exact function has not been elucidated. A yeast two-hybrid screen using ATRAP as bait identified calcium-modulating cyclophilin ligand (CAML) as an ATRAP partner. Yeast two-hybrid and coimmunoprecipitation analysis demonstrated that the N-terminal hydrophilic domain of CAML (amino acids (aa) 1-189) mediates a specific interaction between ATRAP and CAML. Our analysis also showed that aa 40-82 of ATRAP contribute to this interaction. Bioluminescence resonance energy transfer and intracellular colocalization analysis by immunofluorescence in HEK293 cells verified this association within endoplasmic reticulum vesicular structures. Functionally, transcriptional reporter assays and RNA interference ATRAP experiments demonstrated that ATRAP knockdown increased nuclear factor of activated T cells (NFAT) activity. Overexpression of ATRAP decreased Ang II- or CAML-induced NFAT transcriptional activation, whereas an ATRAP-interacting domain of CAML (aa 1-189) sensitized NFAT activation in response to Ang II. These results indicate that CAML is an important signal transducer for the actions of Ang II in regulating the calcineurin-NFAT pathway and suggest that the interaction of CAML with ATRAP may mediate the Ang II actions in vascular physiology.
Guo
Shaodong
S
Cardiovascular Research Laboratories, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Lopez-Ilasaca
Marco
M
Dzau
Victor J
VJ
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000502:physiology*; D000804:Angiotensin II / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D021983:Cyclophilins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015536:Down-Regulation; D004721:Endoplasmic Reticulum / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008024:Ligands; D008954:Models, Biological; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; C089404:CAMLG protein, human; D004268:DNA-Binding Proteins; D008024:Ligands; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D014157:Transcription Factors; D000804:Angiotensin II; D049452:Green Fluorescent Proteins; D021983:Cyclophilins