Abstract
In cardiomyocytes, β1-adrenergic receptor (β1-AR) plays an important role in regulating cardiac functions. Upon continuous ligand stimulation, β1-AR is internalized and mostly recycled back to the plasma membrane (PM). The recycling endosome (RE) is one of the membranous organelles involved in the protein recycling pathway. To determine whether RE is involved in the internalization of β1-AR upon ligand stimulation, we evaluated the localization of β1-AR after stimulation with a β-agonist, isoproterenol (Iso), in β1-AR-transfected COS-1 cells. After 30 min of Iso treatment and cell surface labeling with the appropriate antibodies, β1-AR was internalized from PM and translocated into the perinuclear region, the same location as the transferrin receptor, an RE marker. We then evaluated whether sorting nexin 27 (SNX27) participated in the β1-AR recycling pathway. When β1-AR and SNX27 were coexpressed, β1-AR coimmunoprecipitated with SNX27. In addition, shRNA-mediated silencing of SNX27 compromised β1-AR recycling and enhanced its delivery into lysosome. Overall, β1-AR on PM was internalized into RE upon Iso stimulation and recycled by RE through binding with SNX27 in COS-1 cells.
Highlights
Introduction bAdrenergic receptors (b-ARs) are trimeric G proteincoupled receptors that mediate physiological responses to epinephrine and norepinephrine (Strader et al 1995). b1- and b2-ARs are expressed in the cardiac system and have been shown to play different roles in cardiac function and development (Rohrer et al 1996)
To determine whether recycling endosome (RE) is involved in the internalization of b1-adrenergic receptor (b1-AR) upon ligand stimulation, we evaluated the localization of b1-AR after stimulation with a b-agonist, isoproterenol (Iso), in b1-AR-transfected COS-1 cells
We evaluated whether sorting nexin 27 (SNX27) participated in the b1-AR recycling pathway
Summary
Introduction b-Adrenergic receptors (b-ARs) are trimeric G proteincoupled receptors that mediate physiological responses to epinephrine and norepinephrine (Strader et al 1995). b1- and b2-ARs are expressed in the cardiac system and have been shown to play different roles in cardiac function and development (Rohrer et al 1996). The binding of b-Arrs to the activated b-ARs triggers receptor internalization (Krasel et al 2005). Efficient recycling of b2-AR is dependent on its C-terminal PDZ-domain binding motif (Cao et al 1999). SNX27 might be involved in the functional regulation of many proteins with PDZ-domain binding motifs (Cai et al 2011; Wang et al 2013). It was shown that SNX27 mediates the recycling of PDZ-domain binding motif-containing cargo, b2-AR, by linking to the retromer, a complex of proteins that has been shown to be important in recycling (Temkin et al 2011). We observed that the recycling of b1-AR after Iso-induced internalization is regulated by its binding with SNX27
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