Whether similar mechanisms play a role in
Whether similar mechanisms play a role in adiponectin signal transduction and whether additional proteins are recruited to adiponectin receptors via APPL1 has not been analysed so far. Although it has been clearly established that APPL1 is a downstream adaptor protein in adiponectin signal transduction, the cellular localization of the AdipoR1/APPL1 complex has not been characterized yet. APPL1 is found at the plasma membrane and in early endosomes localized close to the plasma membrane and adiponectin seems not to stimulate APPL1 translocation to the membrane .
AdipoR1 forms a complex with activated protein kinase C1 in hepatocytes Screening of a liver cDNA library with full-length AdipoR1 as bait employing the yeast two-hybrid system identified activated protein kinase C1 (RACK1) as a potential binding protein . The association has been confirmed by co-immunoprecipitation with FLAG-tagged RACK1 and Myc-tagged AdipoR1 co-expressed in COS-7 cells. Recombinant expressed DsRed2-RACK1 displays cytoplasmic localization and GFP-AdipoR1 is predominantly detected at the plasma membrane. Incidental co-localization of AdipoR1 and RACK1 in the cell membrane is strongly enhanced by exogenous adiponectin within 30min of incubation. Knock-down of RACK1 in HepG2 endothelin receptor antagonist impairs adiponectin stimulated glucose uptake . RACK1 is a cytosolic scaffold protein composed of seven WD40 motifs that may associate with distinct proteins . RACK1 is a constituent of the eukaryotic ribosome, and recruits activated protein kinase C thereby stimulating translation . Amongst others an interaction of RACK1 with protein kinase C, Src kinases and the p85 subunit of PI3 kinase and SHP-2 has been confirmed , . Receptors that bind to RACK1 include insulin-like growth factor 1 receptor, type II bone morphogenetic protein receptor and multidrug resistance protein 3 (MDR3/ABCB4) , , . Interestingly, adiponectin and metformin reduce ABCB4 abundance in hepatocytes although different mechanisms seem to be involved , . Whether this may allow enhanced recruitment of RACK1 to AdipoR1, however, needs further investigation.
AdipoR1 forms a complex with protein kinase CK2β subunit in C2C12 and MCF7 cells The N-terminal 136 aminoacids of AdipoR1 have been used to screen a testis cDNA yeast-two-hybrid library and identified protein kinase CK2 β subunit (CK2β) . Immunoprecipitation of the AdipoR1 N-terminus or the whole receptor expressed as EYFP fusion protein in the breast cancer cell line MCF7 has been performed using antibodies against the tag. Coimmunoprecipitation of CK2β with AdipoR1 is only observed after cross-linking the proteins, and therefore, the complex may be either unstable in the experimental settings used or short-lived in vivo . The region of CK2β that binds to AdipoR1 was mapped to the aminoacid-position 113–119 (GHRPPMP) encoding a putative Src homology 3 domain (SH3) . This sequence is conserved in AdipoR2 but complex formation of AdipoR2 and CK2β has not been studied so far. AdipoR1 (EYFP-tagged) and CK2β (HA-tagged) have been expressed in Hek293 cells, and AdipoR1 is predominantly distributed in the cell membrane and CK2β in the cytoplasma. Little co-localization is observed until addition of exogenous adiponectin that stimulates localization of CK2β to the membrane and its co-localization with AdipoR1 . When CK2 activity is blocked by the specific inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benz-imidazole (DMAT) adiponectin-induced phosphorylation of ACC is reduced in C2C12 and MCF7 cells. Inhibition of CK2 with DMAT alone also increases ACC phosphorylation, indicating an adiponectin-independent influence of CK2 in the regulation of ACC phosphorylation . CK2β is the regulatory subunit of the protein serine/threonine kinase CK2. CK2 is involved in cellular processes such as proliferation, apoptosis and differentiation. CK2β has been identified to interact with various proteins like G-alpha subunits to inhibit G protein-coupled receptor activity, with the hemoglobin-haptoglobin scavenger receptor CD163 to stimulate signalling and with p38 MAPK to allosterically activate CK2 in cellular stress situations , , .