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  • What are the in situ effects of multi site CaM

    2024-05-15

    What are the in-situ effects of multi-site CaM associations with AT1R? Kai et al. showed that synthetic peptides corresponding to residues 125–137 (rat sequence) in SMD2, 217–227 in the N-terminal side of SMD3, and 304–316 in SMD4JM inhibit to various degrees AngII-induced GTPase activity of isolated vascular smooth muscle membrane [9]. This indicates that these segments constitute, at least in part, G protein interaction sites for AT1R. Our data now show that the full SMD2 and SMD3, and a.a. 309 – 327 on SMD4JM can all interact with CaM at physiological Ca2+ concentrations. This suggests that CaM binding at these locations may interfere with G protein coupling. Consistently, Zhang et al. have shown in vitro that CaM can compete with peptides corresponding to a.a. 215 – 232 on SMD3 and a.a. 305 – 317 on SMD4JM for interaction with Gβγ subunit [32]. It has been postulated that CaM interaction with SMDs in GPCRs could interfere with G protein coupling via two mechanisms – prevention of G protein-receptor preassociation or promotion of dissociation [25]. Given multi-site interactions between CaM and AT1R with disparate Ca2+ sensitivities, both mechanisms might be in play. For example, with the ability to interact with a.a. 215 – 242 (SMD3) at resting Ca2+ levels, CaM interaction here might prevent Gβγ preassociation at rest. On the other hand, CaM binding at a.a. 125–241 (SMD2) and a.a. 309 – 327 on SMD4JM may promote Gβγ dissociation upon AT1R stimulation. The downstream functional impact of CaM binding to AT1R in living 3965 has not been studied. Inhibition of CaM using W-7 in primary vascular smooth muscle cells virtually abolishes AngII-induced ERK1/2 phosphorylation. Consistently, mutagenesis data indicate that reduction in CaM binding affinity and Ca2+ sensitivity for interaction at each identified domain is associated with significantly reduced AT1R-mediated ERK1/2 phosphorylation. In addition, AngII-induced Ca2+ signals are also significantly reduced in cells expressing mKate2-AT1R with mutant sequence at each domain. In our Ca2+ measurements, cells heterologously expressing a mutant AT1R were recognized by mKate2 fluorescence and selected prior to switching to fura-2 channel, so that the measured signals reflect effect of the intended expressed receptor. Furthermore, the presence of some non-transfected cells in the same microscopic field (absence of mKate2 fluorescence) allowed for comparing Ca2+ signals from cells expressing or not expressing exogenous AT1R in the same experiments. Interestingly, reduced CaM binding at a.a. 125–141 (SMD2) drastically alters the dynamics of AngII-induced Ca2+ signal, from a typical transient into a slow-rising, low amplitude signal. This finding first confirms that signals from the expressed receptors dominated over any endogenous AT1R in this system. However, the finding is surprising considering the lower affinity, dynamic range and Ca2+ sensitivity of CaM interaction with SMD2 than with the other two locations. We do not know the explanation for this. Speculatively, given proximity of the submembrane domains in cells, CaM binding at SMD2 might affect associations at the other domains and thus have impact in cells beyond what biochemical properties of its interaction with SMD2 in isolation would predict. While this is an attractive hypothesis, it is technically challenging at present to test in cells, given that insertion of reporters in an SMD is likely to alter the relative association of binding partners to the remaining SMDs substantially. In conclusion, AT1R possesses up to three CaM-binding domains located at a.a. 125–141 (SMD2), 215–242 (SMD3), and 309–327 (SMD4JM). These domains interact with CaM with disparate affinities and Ca2+ sensitivities in the physiological range of Ca2+ signals in cells. CaM interaction with SMD3 can occur at resting Ca2+ concentration. Functionally, interaction at each domain is important for AngII-stimulated Ca2+ signaling and ERK1/2 phosphorylation.