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    • In this study we have

    2024-03-20

    In this study, we have generated new FRET reporters to screen all submembrane domains of AT1R for interactions with CaM. We identified a new CaM-binding domain on the receptor’s SMD2 (a.a. 125 – 141), confirmed interactions at SMD4JM (a.a. 309 – 327) and characterized the full interacting domain in SMD3. All three domains interact with CaM in a Ca2+-dependent fashion. EC50(Ca2+) values determined for each specific interaction by simultaneous measurements of biosensor responses and free Ca2+ indicate that AT1R-CaM binding at different SMDs can take place at drastically different physiological Ca2+ concentrations in the cell. Mutagenesis studies were used in vitro and in cells to examine the functional impact of CaM binding at the individual sites on AT1R-mediated signaling.
    Materials and Methods
    Results
    Discussion In this study, we have identified several new aspects of the interactions between CaM and AT1R: 1) a new CaM binding site is found spanning a.a. 125–141 on SMD2; 2) full interaction between CaM and SMD3 requires the entire domain, a.a. 215–242; 3) CaM interactions with all three domains require Ca2+ and can occur at different Ca2+ concentrations in the physiological range, with interaction at a.a. 309–327 in SMD4JM having a biphasic sensitivity to Ca2+; and 4) CaM interaction with each binding site is important for AT1R-mediated Ca2+ signaling and ERK1/2 phosphorylation. The identification of a.a. 125–141 on SMD2 as a new binding site for CaM on AT1R is interesting in that the amino chemicals library sequence of SMD2 does not conform to known CaM-binding motifs. This is similar to our identification of SMD1 on GPER as a CaM-binding domain of only 12 residues that can interact with CaM at basal Ca2+ levels [27]. The case of AT1R requiring the entire SMD3 (a.a. 215–242) for full interaction with CaM at this domain is also reminiscent of our observation that fragment a.a. 150–170 of GPER’s SMD2 binds CaM, but with a substantially lower affinity than the full-length SMD2, a.a. 150–175, indicating an important role of the basic patch a.a. 170–175 in this process [27]. In the case of AT1R’s SMD3, a.a. 233 – 242 contains a high percentage of hydrophobic and charged residues, which apparently contribute significantly to CaM binding, adding a 3-fold increase in binding affinity and doubling Ca2+ sensitivity for the interaction with the full SMD3, a.a. 215 – 242, vs the previously reported segment, a.a. 215 – 232. These findings further support the value of using FRET biosensors to screen for CaM-binding sites in GPCRs. Simultaneous measurements of BSAT1RX-CaM interaction and corresponding free Ca2+ concentrations indicate that the interactions between CaM and the three identified domains on AT1R can all occur during a typical cytoplasmic Ca2+ signal in VSMCs. With disparate affinities and Ca2+ sensitivities, CaM binding may occur at different submembrane domains at different levels of free Ca2+. SMD3-CaM interaction has an EC50(Ca2+) value of 150 nM (Fig. 5 and Table 4) and at 50 nM free Ca2+, there is significant binding at this domain in vitro. These values are well within the resting range of cytoplasmic Ca2+ in VSMCs, suggesting that AT1R-CaM interaction can occur in basal conditions in cells. Considering a competitive environment for CaM among CaM-dependent proteins due to limiting CaM [14], [23], [24], [25], CaM interactions with EC50(Ca2+) values in the range of basal Ca2+ levels are more likely to occur since fewer target proteins bind CaM at this level of Ca2+ compared to during a large Ca2+ signals. For the remaining domains, EC50(Ca2+) values of 0.31, 3.18 and 4.1 μM indicate that CaM-SMD4JMSp1, CaM-SMD4JMSp2 and CaM-SMD2 complexes, respectively, are likely to take place at different points during the time course of a typical agonist-stimulated response. Based on Ca2+ titration curve, full association between CaM and a.a. 125–141 on SMD2 requires ∼10 μM free Ca2+. This is attainable in cells, as sub-PM Ca2+ in VSMCs reaches 45 μM and remains above 5 μM throughout the time course of the vasopressin-induced Ca2+ signal [16].