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Characterization and Functional Effects of Multi‐site Interactions between calmodulin and the angiotensin II receptor type 1A
Author(s) -
Clements Robert,
Ehlers Kevin,
VerMeer Mark,
Tran Kim
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.lb536
Subject(s) - calmodulin , thapsigargin , angiotensin ii , chemistry , intracellular , biophysics , microbiology and biotechnology , receptor , c2 domain , biochemistry , biology , membrane , enzyme
The angiotensin II receptor type 1A (AT 1A R) is responsible for many effects of angiotensin II (AngII). Calmodulin (CaM) is essential for many cell functions due to its interactions with many proteins. CaM has previously been demonstrated to interact with AT 1A R at a.a. 305 – 327 in the fourth submembrane domain (SMD4) (Thomas WG et al., FEBS Lett. 1999;455:367–371) and a.a. 214 – 231 in the third submembrane domain (Zhang R et al., PLOS One 2013;8:e65266). The functional effects of CaM interaction with AT 1A R, however, remains incompletely understood. Additionally, the interaction domains are not completely identified and characterized. We have observed that AT1R coimmunoprecipitates with CaM in vascular smooth muscle under resting conditions, association that is enhanced by stimulation with AngII or thapsigargin. Using novel FRET biosensors with EYFP‐ECFP pair flanking each of AT 1A R's submembrane domains, we have identified a novel CaM‐binding domain in the submembrane domain 2 and found that full interaction between CaM and SMD3 requires a significantly longer sequence than previously reported. All three domains interact with CaM in a Ca 2+ ‐dependent fashion, with drastically different affinities. Simultaneous determinations of biosensor responses and free Ca 2+ concentrations showed that these interactions take place at significantly different Ca 2+ sensitivities in the physiological range of intracellular Ca 2+ . Notably, SMD3 interacts with CaM with a Ca 2+ sensitivity that falls in the range of resting intracellular Ca 2+ concentration. Cells heterologously expressing full‐length AT 1A R containing mutations that reduced CaM interactions at individual CaM binding domains on SMD2, SMD3 or SMD4, exhibit ~ 1.8‐, 2‐ or 3‐fold reductions in AngII‐stimulated ERK1/2 phosphorylation, respectively. These data demonstrate that CaM is involved in AngII signaling via direct interactions with multiple domains in AT1R and allow prediction of interactions between CaM and individual SMDs under distinct physiological scenarios. Support or Funding Information Iowa Osteopathic and Educational Research Funds National Institutes of Health Grant HL‐112184