Phosphoinositide 3-kinase regulates β2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/β-arrestin complex

Internalization of β-adrenergic receptors (βARs) occurs by the sequential binding of β-arrestin, the clathrin adaptor AP-2, and clathrin. D-3 phosphoinositides, generated by the action of phosphoinositide 3-kinase (PI3K) may regulate the endocytic process; however, the precise molecular mechanism is unknown. Here we demonstrate that βARKinase1 directly interacts with the PIK domain of PI3K to form a cytosolic complex. Overexpression of the PIK domain displaces endogenous PI3K from βARK1 and prevents βARK1-mediated translocation of PI3K to activated β2ARs. Furthermore, disruption of the βARK1/PI3K interaction inhibits agonist-stimulated AP-2 adaptor protein recruitment to the β2AR and receptor endocytosis without affecting the internalization of other clathrin dependent processes such as internalization of the transferrin receptor. In contrast, AP-2 recruitment is enhanced in the presence of D-3 phospholipids, and receptor internalization is blocked in presence of the specific phosphatidylinositol-3,4,5-trisphosphate lipid phosphatase PTEN. These findings provide a molecular mechanism for the agonist-dependent recruitment of PI3K to βARs, and support a role for the localized generation of D-3 phosphoinositides in regulating the recruitment of the receptor/cargo to clathrin-coated pits

Penerapan Metode Eksperimen untuk Meningkatkan Konsep Dasar Sains pada Anak Didik Kelompok A Tk Pkk Suruhwadang Kecamatan Kademangan Kabupaten Blitar

Tujuan penelitian ini adalah untuk memperoleh tentang kemampuan kognitif anak dalamhal konsep dasar sains dengan menggunakan metode eksperimen pada anak didik kelompokA TK PKK Suruhwadang sebelum dan sesudah dilakukan tindakan. Melakukan tindakanberupa penerapan metode eksperimen untuk meningkatkan kemampuan kognitif dalamkonsep dasar sains pada anak didik kelompok A TK PKK Suruhwadang. Mengetahui adatidaknya perbedaan kemampuan konsep dasar sains dengan menggunakan metodeeksperimen pada anak didik kelompok A TK PKK Suruhwadang antara sebelum dan setelahdilakukan tindakan. Rumusan masalah pada penitilian ini adalah apakah metode eksperimendapat meningkatkan kemampuan pemahaman konsep dasar sains pada anak didik kelompokA TK PKK Suruhwadang Kecamatan Kademangan Kabupaten Blitar. Untuk menjawabrumusan masalah digunakan jenis penelitian tindakan kelas (PTK) dengan model Kemmisdan Taggart melalui empat tahapan yaitu tahap perencanaan , pelaksanaan, observasi danrefleksiyang dilalui dengan dua siklus. Teknik pengumpulan data menggunakan teknikobservasi dan dokumentasi. Adapun instrumen yang digunakan adalah lembar observasikegiatan anak dan lembar observasi pembelajaran oleh guru.Hasil penelitian menunjukanbahwa kemampuan kognitif anak kelompok A pada konsep dasar sain pada pra penelitianmenunjukkan prosentase 56.25%. Setelah pelaksanaan siklus I tentang bidang kemampuankognitif pada konsep dasar sains menunjukkan 59% mengalami peningkatan .Setelahpelaksanaan siklus ke II naik menjadi 83%. Hal ini menunjukkan pelaksanaan siklus ke IItelah mencapai kriteria ketuntasan dan membuktikan bahwa dengan metode eksperimendapat meningkatkan kemampuan kognitif dalam konsep dasar sains

Inhibition of receptor-localized PI3K preserves cardiac β-adrenergic receptor function and ameliorates pressure overload heart failure

β-Adrenergic receptor (βAR) downregulation and desensitization are hallmarks of the failing heart. However, whether abnormalities in βAR function are mechanistically linked to the cause of heart failure is not known. We hypothesized that downregulation of cardiac βARs can be prevented through inhibition of PI3K activity within the receptor complex, because PI3K is necessary for βAR internalization. Here we show that in genetically modified mice, disrupting the recruitment of PI3K to agonist-activated βARs in vivo prevents receptor downregulation in response to chronic catecholamine administration and ameliorates the development of heart failure with pressure overload. Disruption of PI3K/βAR colocalization is required to preserve βAR signaling, since deletion of a single PI3K isoform (PI3Kγ knockout) is insufficient to prevent the recruitment of other PI3K isoforms and subsequent βAR downregulation with catecholamine stress. These data demonstrate a specific role for receptor-localized PI3K in the regulation of βAR turnover and show that abnormalities in βAR function are associated with the development of heart failure. Thus, a strategy that blocks the membrane translocation of PI3K and leads to the inhibition of βAR-localized PI3K activity represents a novel therapeutic approach to restore normal βAR signaling and preserve cardiac function in the pressure overloaded failing heart

Cardiac hypertrophy and altered {beta}-adrenergic signaling in transgenic mice expressing the amino terminus of {beta}ARK1

The G protein-coupled receptor (GPCR) kinase, {beta}-adrenergic receptor ({beta}AR) kinase ({beta}ARK1) is elevated in heart failure, however its role is not fully understood. {beta}ARK1 contains several domains capable of protein-protein interactions that may play critical roles in the regulation of GPCR signaling. In this study, we developed a novel line of transgenic mice that express an amino-terminal peptide of {beta}ARK1, comprised of amino acid residues 50-145 ({beta}ARKnt), in the heart, to determine if this domain has any functional significance in vivo. Surprisingly, the {beta}ARKnt transgenic mice presented with cardiac hypertrophy. Our data suggest that the phenotype was driven via an enhanced {beta}AR system, as {beta}ARKnt mice had elevated cardiac {beta}AR density. Moreover, administration of a {beta}AR antagonist reversed hypertrophy in these mice. Interestingly, signaling through the {beta}AR in response to agonist stimulation was not enhanced in these mice. Thus, the amino terminus of {beta}ARK1 appears critical for normal {beta}AR regulation in vivo, and further supports the hypothesis that {beta}ARK1 plays a key role in normal and compromised cardiac GPCR signaling