MECHANISMS INVOLVED IN ISOFORM SPECIFIC BETA ARRESTIN INTERACTION AND PHOSPHORYLATION BY DELTA CAMKII

Ca²⁺/calmodulin-dependent kinase II (CaMKII) activation during chronic β adrenergic receptor 1 (βADR1) stimulation contributes to cardiac hypertrophy in heart failure (HF). β arrestins 1 and 2, are non-visual arrestins classically linked to G protein-coupled receptor (GPCR) recruitment during receptor desensitization. β arrestins also play a critical role in multiple signaling cascades, as they localize a number of signaling effectors, including CaMKII. The goal of our study is to determine if δ_CCaMKII, the predominant cytoplasmic isoform CaMKII in the heart, directly interacts with β arrestins and whether specific regions of β arrestins target δ_CCaMKII and whether β arrestins δ_CCaMKII substrates. Using SPOTS peptide arrays, we mapped four potential sites (S193, S196, S202, T227) to the C-lobe of recombinant b arrestins. Using GST tagged β arrestins 1 and 2 as bait proteins, we identified that naïve δ_CCaMKII fails to bind β arrestin 2, but Thr287 autophosphorylation enhances binding and this δ_CCaMKII interaction is regulated by the β arrestin 2 C-tail. β arrestin 1 failed to interact with δ_CCaMKII (naïve or Thr287 autophosphorylated). In addition to interacting with autophosphorylated δ_CCaMKII, we also report that β arrestin 2 can interact with naïve δ_CCaMKII when the C-tail is truncated or displaced using a βADR1 C-tail derived phosphomimetic peptide. While Ca²⁺/CaM inhibited autophosphorylated δ_CCaMKII binding to β arrestin 2, it did not inhibit the binding of naïve δ_CCaMKII, suggesting the potential for Ca²⁺ signaling to both promote δ_CCaMKII activation and reduce δ_CCaMKII targeting to β arrestins. Using SPOTs peptide arrays, we identified three high-affinity binding sites on β arrestin 2 and one on β arrestin 1 targeting fluorescently-labeled Thr287 autophosphorylated δ_CCaMKII_Alexa790. Different binding patterns for naïve versus activated δ_CCaMKII were observed, suggesting distinct binding modes for inactive versus activated CaMKII. A soluble version of one CaMKII interactor peptide from b arrestin 2 (190LRP) induced β arrestin 1 and β arrestin 2 binding to naïve δ_CCaMKII. Our research reveals a complex landscape whereby β arrestins differentially interact with naïve and activated δ_CCaMKII, a process we hypothesize to contribute to the maladaptive changes in HF.