br Endocytosis of AdipoR Surface receptor activity may

Endocytosis of AdipoR1 Surface receptor activity may be shut down by endocytic uptake and subsequent lysosomal degradation of the proteins [20]. Clathrin-dependent endocytosis (CDE) has been extensively studied and internalization from the plasma membrane is mediated through clathrin-coated pits and vesicles, and ubiquitination-dependent delivery into endosomal vesicles [24]. Cells may internalize the equivalent of their cell surface one to five times per hour [25], and therefore, the removal of plasma membrane proteins and lipids is counterbalanced by recycling pathways. Endocytic receptor uptake is either stimulated by ligand binding or is a constitutive and ligand-independent process. Different endocytotic pathways may be involved in the internalization of a specific receptor protein with distinct biological responses. For instance uptake of transforming growth factor β (TGF-β) receptor via CDE promotes signalling whereas caveolin-mediated uptake regulates receptor turnover [26]. AdipoR1 and AdipoR2 are predicted to be located at the plasma membrane [10]. Besides residing at the plasma membrane AdipoR1 and AdipoR2 are also distributed in the cytoplasma with AdipoR1 being located in cytoplasmic puncta [27]. Overexpression of C-terminally myc-tagged AdipoR1 in Hek293 Necrosulfonamide was employed to study protein internalization which starts within 5min and is completed 30–60min later. Recombinant full-length adiponectin is taken up by cells as has been demonstrated for hepatocytes, epithelial cells and HeLa cells [27], [28], [29]. Globular adiponectin, the globular C1q domain of adiponectin generated by proteolysis of the full-length protein is also internalized by HeLa cells [27]. In contrast baculovirus-produced trimeric adiponectin [30] is not incorporated by hepatocytes (own unpublished observation). Adiponectin colocalizes with AdipoR1 indicating similar trafficking routes of both proteins (Fig. 1). Exogenously provided adiponectin does not influence endocytosis of AdipoR1 suggesting ligand-independent internalization. Colocalization of endocytosed adiponectin and AdipoR1 with transferrin and the transferrin receptor surmise clathrin-mediated uptake of adiponectin and AdipoR1. This is further proven by overexpression of Eps15 mutants, potassium depletion and hypertonic medium that all inhibit CDE and AdipoR1 internalization [27]. The small GTPase Rab5 plays an important role in the formation and dynamics of early/sorting endosomes [31]. Colocalization of AdipoR1 and Rab5 and blockage of AdipoR1 endocytosis by a dominant-negative Rab5 mutant confirms a critical role of Rab5 in AdipoR1 uptake [27]. Blocking endocytosis of AdipoR1 enhances adiponectin stimulated phosphorylation of AMPK and acetyl-CoA carboxylase (ACC). Whether this is explained by higher plasma membrane levels of AdipoR1 has not been evaluated so far. In addition basal levels of phosphorylated AMPK and ACC are elevated when endocytosis of AdipoR1 is inhibited [27] (Fig. 1). Overexpression of AdipoR1 in yeast is sufficient to stimulate ligand-independent signalling [32], and additional studies have to prove if similar mechanisms are responsible for this effect. In conclusion, clathrin-dependent endocytosis of AdipoR1 seems to downmodulate adiponectin signalling.
APPL1 stimulates AdipoR1 signalling Recently, APPL1 has been identified as an AdipoR1 and AdipoR2 binding protein. The N-terminal amino acids (4–142) of AdipoR1 interact with the phosphotyrosine binding domain (PTB) of APPL1 whereas its pleckstrin homology domain and the Bin-amphiphysin-Rvs (BAR) domain are dispensable for this association. The formation of the AdipoR1 APPL1 complex is enhanced by adiponectin [33]. APPL1 is highly abundant in mouse skeletal muscle and differentiated C2C12 myotubes and moderately expressed in hepatocytes [33]. Overexpression of APPL1 in C2C12 myotubes is associated with increased basal and adiponectin-stimulated phosphorylation of AMPK and p38 MAPK whereas small interference RNA-mediated suppression of APPL1 impairs adiponectin-stimulated phosphorylation of AMPK, p38 MAPK and ACC [33]. Knock-down of APPL1 is even associated with a reduced expression of AMPK protein indicating that APPL1 may be a crucial adaptor protein in metabolic control besides its role in adiponectin signalling [33].