Perturbation of the interactions of calmodulin with GRK5 using a natural product chemical probe

Abstract

G protein-coupled receptor (GPCR) kinases (GRKs) are responsible for initiating desensitization of activated GPCRs. GRK5 is potently inhibited by the calcium-sensing protein calmodulin (CaM), which leads to nuclear translocation of GRK5 and promotion of cardiac hypertrophy. Herein, we report the architecture of the Ca2+·CaM–GRK5 complex determined by small-angle X-ray scattering and negative-stain electron microscopy. Ca2+·CaM binds primarily to the small lobe of the kinase domain of GRK5 near elements critical for receptor interaction and membrane association, thereby inhibiting receptor phosphorylation while activating the kinase for phosphorylation of soluble substrates. To define the role of each lobe of Ca2+·CaM, we utilized the natural product malbrancheamide as a chemical probe to show that the C-terminal lobe of Ca2+·CaM regulates membrane binding while the N-terminal lobe regulates receptor phosphorylation and kinase domain activation. In cells, malbrancheamide attenuated GRK5 nuclear translocation and effectively blocked the hypertrophic response, demonstrating the utility of this natural product and its derivatives in probing Ca2+·CaM-dependent hypertrophy.

Antagonistic Roles of GRK2 and GRK5 in Cardiac Aldosterone Signaling Reveal GRK5-Mediated Cardioprotection via Mineralocorticoid Receptor Inhibition

“…To delineate the signaling mechanism(s) underlying stimulation of GRK5-mediated phosphorylation of the cardiac MR by the β 2 AR, we first examined the subcellular localization of the GRK5-MR interaction in H9c2 cardiac myocytes. Since both the MR and GRK5 can localize in both the cytoplasm and the nucleus [5,22,23], one important question arising from our present findings is which subcellular fraction these two proteins interact with each other in. To this end, we subjected H9c2 cell lysates to subcellular fractionation and we performed co-IP experiments in cytosolic and nuclear fraction extracts.…”

“…In support of this, a recent study reported that protein kinase A (PKA), robustly activated by the cardiac β 1 AR, inhibits β 2 AR's ability to stimulate Ca 2+ -CaM signaling via ERKs and IP 3 in the caveolae of atrial myocytes [37]. It is thus quite plausible that the β 1 AR antagonizes the β 2 AR-induced, Ca 2+ -dependent GRK5 cytoplasmic/nuclear translocation in cardiac myocytes [22]. Of note, although it normally does not couple to G q proteins, the β 2 AR has been reported to directly activate PLCβ, eliciting intracellular IP 3 -dependent Ca 2+ mobilization/signaling [38].…”

“…In the present study, we have uncovered a new, noncanonical, and advantageous function of cardiac GRK5: inhibitory phosphorylation of the MR (Figure 7). This GRK5 effect is β 2 AR-dependent and proceeds through a noncanonical PLCβ-Ca 2+ -CaM signaling pathway (Figure 7), resulting in GRK5 translocation from the plasma membrane to the cytoplasm [22]. The MR has long been established as an important molecular culprit in heart disease progression [27].…”

“…Notably, the MR was recently shown to promote heart failure by activating GRK2-dependent apoptosis and GRK5 nuclear accumulation-dependent hypertrophy in transgenic mouse hearts in vivo [30]. These noncanonical, deleterious effects of the two GRKs were mediated by MR-induced, c-Src kinase-dependent cardiac angiotensin II type 1 receptor (AT 1 R) transactivation [22,30].…”

“…Given that GRK5 is normally plasma membrane-anchored, thanks to its constitutive interaction with membrane phospholipids [9], an important question pertains to the mechanism of its translocation to the cytoplasm, wherein it phosphorylates the MR. Recent studies have revealed that the ubiquitous Ca 2+ -binding protein CaM, known to inhibit GRK5 membrane localization and activity toward GPCR phosphorylation [24], can markedly increase GRK5 activity towards non-GPCR (noncanonical) substrates as well as can induce GRK5 translocation to the nucleus [22]. The latter process has been reported to mediate GRK5-depedendent cardiac hypertrophy [23].…”