Localization of novel adiponectin receptor constructs

Adiponectin is one of the most abundant fat-derived hormones involved in a multitude of metabolism pathways. The receptors AdipoR1 and AdipoR2 of this metabolically active protein have been identified recently. AdipoR1 and AdipoR2 are most abundantly expressed in the skeletal muscle and in the liver, respectively. It has been postulated that although they both consist of seven transmembrane helices, they are distinct from other G protein-coupled receptors (GPCRs). We cloned both receptors as fusion proteins with enhanced yellow fluorescent protein (YFP) to determine their localization and orientation in the cell membrane. By confocal microscopy and immune staining we demonstrated that both receptor-YFP-fusion proteins are integral membrane proteins with the predicted topology--an intracellular N-terminus and an extracellular C-terminus. In parallel, comparative experiments were performed with the NPY Y2-receptor, a classical rhodopsin-like GPCR.     

Expression profiles of adiponectin receptors in mouse embryos

Adiponectin is a protein secreted from adipocytes and it plays an important endocrine role in glucose and lipid homeostasis. A reverse correlation between plasma adiponectin concentrations and insulin resistance has been established in both animals and humans. Adiponectin exerts its function by interacting with membrane receptors, including AdipoR1 and AdipoR2. We investigated the expression pattern of these two adiponectin receptors in mouse embryos. At stages E12.5 and E15.5, both AdipoR1 and AdipoR2 are highly expressed in the nervous system including the trigeminal ganglion, glossopharyngeal ganglion and dorsal root ganglia. AdipoR1 is highly expressed in many tissues derived from primitive gut, including the lung, liver, pancreas and small intestines. Generally, the expression level of AdipoR2 is weaker and more restricted than AdipoR1 in most of the tissues. In addition, AdipoR1 expression can be found in heart, vertebrate, developing bones and cartilage, and many other tissues. This study reveals that AdipoR1 and AdipoR2 have differential but overlapping expression profiles during mouse development.     

ERp46 binds to AdipoR1, but not AdipoR2, and modulates adiponectin signalling

The pleiotropic effects of the insulin-sensitizing adipokine adiponectin are mediated, at least in part, by two seven-transmembrane domain receptors AdipoR1 and AdipoR2. Recent reports indicate a role for AdipoR-binding proteins, namely APPL1, RACK1 and CK2β, in proximal signal transduction events. Here we demonstrate that endoplasmic reticulum protein 46 (ERp46) interacts specifically with AdipoR1 and provide evidence that ERp46 modulates adiponectin signalling. Co-immunoprecipitation followed by mass spectrometry identified ERp46 as an AdipoR1-, but not AdipoR2-, interacting protein. Analysis of truncated constructs and GST-fusion proteins revealed the interaction was mediated by the cytoplasmic, N-terminal residues (1-70) of AdipoR1. Indirect immunofluorescence microscopy and subcellular fractionation studies demonstrated that ERp46 was present in the ER and the plasma membrane (PM). Transient knockdown of ERp46 increased the levels of AdipoR1, and AdipoR2, at the PM and this correlated with increased adiponectin-stimulated phosphorylation of AMPK. In contrast, adiponectin-stimulated phosphorylation of p38MAPK was reduced following ERp46 knockdown. Collectively these results establish ERp46 as the first AdipoR1-specific interacting protein and suggest a role for ERp46 in adiponectin receptor biology and adiponectin signalling.     

Adiponectin receptor binding proteins - recent advances in elucidating adiponectin signalling pathways

Adiponectin whose systemic levels are reduced in obesity-related diseases ameliorates insulin sensitivity and regulates biological processes like apoptosis, proliferation, migration and inflammation. Adiponectin binds to adiponectin receptors, AdipoR1 and AdipoR2, which are ubiquitously expressed. Clathrin-dependent endocytosis of AdipoR1 and adiponectin has been demonstrated to modulate adiponectin bioactivity. Recently, APPL1 has been identified as an AdipoR1 and AdipoR2 binding protein. Furthermore, activated protein kinase C1, endoplasmic reticulum protein 46 and protein kinase CK2β subunit form a complex with AdipoR1. This review summarizes recent studies exploiting heterologous expression of adiponectin receptors in yeast, and the type and function of the recently described adiponectin receptor associated proteins.     

Expression and Biologic Significance of Adiponectin Receptors in Papillary Thyroid Carcinoma

Obesity is associated with a higher incidence of thyroid cancer. Adiponectin is one of the most abundant adipokines with a pleiotropic role in metabolism and in the development and progression of cancer. It has been shown that circulating adiponectin level is inversely associated with the risk of thyroid cancer. This study aimed to investigate the possible association between the expression of adiponectin receptors (AdipoR1 and AdipoR2) and clinicopathological variables in papillary thyroid cancer. We found that protein levels of AdipoR1 and AdipoR2 were increased in some thyroid cancer specimens compared with adjacent normal thyroid tissues. Thyroid cancer cells expressed AdipoR1 and AdipoR2, which were attenuated by histone deacetylase inhibitors valproic acid and trichostatin A. Adiponectin stimulated AMP-activated protein kinase phosphorylation in thyroid cancer cells. We further determined the expression of AdipoR1 and AdipoR2 by immunohistochemical staining in primary tumor samples and metastatic lymph nodes. AdipoR1 was expressed in 27 % of primary tumors and AdipoR2 in 47 %. Negative expression of both adiponectin receptors was significantly associated with extrathyroidal invasion, multicentricity, and higher TNM stage. There was a trend toward decreased disease-free survival in patients with negative tumor expression of AdipoR1 and AdipoR2 (log-rank P = 0.051). Collectively, overexpression of adiponectin receptors was observed in some tumor tissues of papillary thyroid cancer and was associated with a better prognosis.     

A Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signaling

Adiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc) activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc) in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p). The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides’ ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA) signaling and AMP activated protein kinase (AMPK) phosphorylation in S. cerevisiae, which are homologous to important mammalian adiponectin-AdipoR1 signaling pathways. This system should facilitate the development of therapeutic inventions targeting adiponectin and/or AdipoR physiology.     

Endocytosis of adiponectin receptor 1 through a clathrin- and Rab5-dependent pathway

In eukaryotic cells, receptor endocytosis is a key event regulating signaling transduction. Adiponectin receptors belong to a new receptor family that is distinct from G-protein-coupled receptors and has critical roles in the pathogenesis of diabetes and metabolic syndrome. Here, we analyzed the endocytosis of adiponectin and adiponectin receptor 1 (AdipoR1) and found that they are both internalized into transferrin-positive compartments that follow similar traffic routes. Blocking clathrin-mediated endocytosis by expressing Eps15 mutants or depleting K(+) trapped AdipoR1 at the plasma membrane, and K(+) depletion abolished adiponectin internalization, indicating that the endocytosis of AdipoR1 and adiponectin is clathrin-dependent. Depletion of K(+) and overexpression of Eps15 mutants enhance adiponectin-stimulated AMP-activated protein kinase phosphorylation, suggesting that the endocytosis of AdipoR1 might downregulate adiponectin signaling. In addition, AdipoR1 colocalizes with the small GTPase Rab5, and a dominant negative Rab5 abrogates AdipoR1 endocytosis. These data indicate that AdipoR1 is internalized through a clathrin- and Rab5-dependent pathway and that endocytosis may play a role in the regulation of adiponectin signaling.     

APPL1 binds to adiponectin receptors and mediates adiponectin signalling and function

Adiponectin, also known as Acrp30, is an adipose tissue-derived hormone with anti-atherogenic, anti-diabetic and insulin sensitizing properties. Two seven-transmembrane domain-containing proteins, AdipoR1 and AdipoR2, have recently been identified as adiponectin receptors, yet signalling events downstream of these receptors remain poorly defined. By using the cytoplasmic domain of AdipoR1 as bait, we screened a yeast two-hybrid cDNA library derived from human fetal brain. This screening led to the identification of a phosphotyrosine binding domain and a pleckstrin homology domain-containing adaptor protein, APPL1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding (PTB) domain and leucine zipper motif). APPL1 interacts with adiponectin receptors in mammalian cells and the interaction is stimulated by adiponectin. Overexpression of APPL1 increases, and suppression of APPL1 level reduces, adiponectin signalling and adiponectin-mediated downstream events (such as lipid oxidation, glucose uptake and the membrane translocation of glucose transport 4 (GLUT4)). Adiponectin stimulates the interaction between APPL1 and Rab5 (a small GTPase) interaction, leading to increased GLUT4 membrane translocation. APPL1 also acts as a critical regulator of the crosstalk between adiponectin signalling and insulin signalling pathways. These results demonstrate a key function for APPL1 in adiponectin signalling and provide a molecular mechanism for the insulin sensitizing function of adiponectin.     

Regulation of adiponectin receptor 1 in human hepatocytes by agonists of nuclear receptors

The adiponectin receptors AdipoR1 and AdipoR2 have been identified to mediate the insulin-sensitizing effects of adiponectin. Although AdipoR2 was suggested to be the main receptor for this adipokine in hepatocytes, AdipoR1 protein is highly abundant in primary human hepatocytes and hepatocytic cell lines. Nuclear receptors are main regulators of lipid metabolism and activation of peroxisome proliferator-activated receptor alpha and gamma, retinoid X receptor (RXR), and liver X receptor (LXR) by specific ligands may influence AdipoR1 abundance. AdipoR1 protein is neither altered by RXR or LXR agonists nor by pioglitazone. In contrast, fenofibric acid reduces AdipoR1 whereas hepatotoxic troglitazone upregulates AdipoR1 protein in HepG2 cells. Taken together this work shows for the first time that AdipoR1 protein is expressed in human hepatocytes but that it is not a direct target gene of nuclear receptors. Elevated AdipoR1 induced by hepatotoxic troglitazone may indicate a role of this receptor in adiponectin-mediated beneficial effects in liver damage.     

Expression of adiponectin receptors in human macrophages and regulation by agonists of the nuclear receptors PPARalpha, PPARgamma, and LXR

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors expressed in macrophages where they control cholesterol homeostasis and inflammation. In an attempt to identify new PPARalpha and PPARgamma target genes in macrophages, a DNA array-based global gene expression profiling experiment was performed on human primary macrophages treated with specific PPARalpha and PPARgamma agonists. Surprisingly, AdipoR2, one of the two recently identified receptors for adiponectin, an adipocyte-specific secreted hormone with anti-diabetic and anti-atherogenic activities, was found to be induced by both PPARalpha and PPARgamma. AdipoR2 induction by PPARalpha and PPARgamma in primary and THP-1 macrophages was confirmed by Q-PCR analysis. Interestingly, treatment with a synthetic LXR agonist induced the expression of both AdipoR1 and AdipoR2. Furthermore, co-incubation with a PPARalpha ligand and adiponectin resulted in an additive effect on the reduction of macrophage cholesteryl ester content. Finally, AdipoR1 and AdipoR2 are both present in human atherosclerotic lesions. Moreover, AdipoR1 is more abundant than AdipoR2 in monocytes and its expression decreases upon differentiation into macrophages, whereas AdipoR2 remains constant. In conclusion, AdipoR1 and AdipoR2 are expressed in human atherosclerotic lesions and macrophages and can be modulated by PPAR and LXR ligands, thus identifying a mechanism of crosstalk between adiponectin and these nuclear receptor signaling pathways.     

Receptor for activated C-kinase 1, a novel binding partner of adiponectin receptor 1

Adiponectin is an adipose tissue derived hormone with anti-diabetic and insulin-sensitizing properties. Two adiponectin receptors, AdipoR1 and AdipoR2, have recently been identified, yet the signaling pathways triggered through adiponectin receptors remain to be elucidated. Using a yeast two-hybrid screen, we identified an adaptor protein, receptor for activated protein kinase C1 (RACK1), as an interacting partner of human AdipoR1. RACK1 was confirmed to interact with AdipoR1 by co-immunoprecipitation and co-localization analysis in mammalian cells. The interaction was enhanced by adiponectin stimulation. In addition, the knockdown of RACK1 by RNA interference inhibited adiponectin-stimulated glucose uptake in HepG2 cells. These results suggest that RACK1 may act as a key bridging factor in adiponectin signaling transduction through interacting with AdipoR1.     

ATF3 negatively regulates adiponectin receptor 1 expression

Adiponectin is an adipocyte-derived hormone that has antidiabetic and antiatherogenic effects through two membrane receptors, adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2). Although it has been reported that the expression of AdipoR1 and AdipoR2 is regulated under physiological and pathophysiological states, their regulation is largely unknown. Previously, we demonstrated that endoplasmic reticulum (ER) stress or obesity-inducible ATF3 negatively regulates the expression of adiponectin and AdipoR2. Here, we investigated the regulation of another adiponectin receptor, AdipoR1 by ATF3, to determine if ATF3 may contribute to impairment of adiponectin signaling by repressing the expression of both adiponectin and adiponectin receptors. We found that treatment with thapsigargin, a stimulator of ATF3 expression as an inducer of ER stress, decreased AdipoR1 expression in insulin-sensitive cells (HepG2, C2C12) and insulin secreting cells (MIN6N8). Furthermore, overexpression of lentivirus carrying-ATF3 decreased AdipoR1 expression in those cells, demonstrating that ATF3 downregulates AdipoR1 expression. Next, we investigated the effects of ATF3 on human AdipoR1 promoter activity and identified an ATF3-responsive region in the promoter. Both thapsigargin treatment and ATF3 expression repressed AdipoR1 promoter activity. Transfection studies using mutant constructs containing 5′-deletions in the human AdipoR1 promoter revealed that putative ATF/CRE site is located between the −248 and −224, TGACGCGG. Chromatin immunoprecipitation assay demonstrated that ATF3 directly binds to human AdipoR1 promoter spanning from −248 to −224. Finally, deletion of the putative ATF/CRE site abrogated ATF3-mediated transrepression of the AdipoR1 promoter. Importantly, ATF3 expression was increased in hyperglycemia or TNF-α-treated C2C12 cells in which AdipoR1 expression was decreased, suggesting that ATF3 may contribute to downregulation of AdipoR1 by hyperglycemia and TNF-α. Collectively, these results demonstrate that ATF3 negatively regulates human AdipoR1 expression via binding to an ATF3-responsive region in the promoter, which plays an important role in attenuation of adiponectin signaling and induction of insulin resistance.     

APPL1: role in adiponectin signaling and beyond

Adiponectin, an adipokine secreted by the white adipose tissue, plays an important role in regulating glucose and lipid metabolism and controlling energy homeostasis in insulin-sensitive tissues. A decrease in the circulating level of adiponectin has been linked to insulin resistance, type 2 diabetes, atherosclerosis, and metabolic syndrome. Adiponectin exerts its effects through two membrane receptors, AdipoR1 and AdipoR2. APPL1 is the first identified protein that interacts directly with adiponectin receptors. APPL1 is an adaptor protein with multiple functional domains, the Bin1/amphiphysin/rvs167, pleckstrin homology, and phosphotyrosine binding domains. The PTB domain of APPL1 interacts directly with the intracellular region of adiponectin receptors. Through this interaction, APPL1 mediates adiponectin signaling and its effects on metabolism. APPL1 also functions in insulin-signaling pathway and is an important mediator of adiponectin-dependent insulin sensitization in skeletal muscle. Adiponectin signaling through APPL1 is necessary to exert its anti-inflammatory and cytoprotective effects on endothelial cells. APPL1 also acts as a mediator of other signaling pathways by interacting directly with membrane receptors or signaling proteins, thereby playing critical roles in cell proliferation, apoptosis, cell survival, endosomal trafficking, and chromatin remodeling. This review focuses mainly on our current understanding of adiponectin signaling in various tissues, the role of APPL1 in mediating adiponectin signaling, and also its role in the cross-talk between adiponectin/insulin-signaling pathways.     

Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions

Adiponectin plays a central role as an antidiabetic and antiatherogenic adipokine. AdipoR1 and AdipoR2 serve as receptors for adiponectin in vitro, and their reduction in obesity seems to be correlated with reduced adiponectin sensitivity. Here we show that adenovirus-mediated expression of AdipoR1 and R2 in the liver of Lepr(-/-) mice increased AMP-activated protein kinase (AMPK) activation and peroxisome proliferator-activated receptor (PPAR)-alpha signaling pathways, respectively. Activation of AMPK reduced gluconeogenesis, whereas expression of the receptors in both cases increased fatty acid oxidation and lead to an amelioration of diabetes. Alternatively, targeted disruption of AdipoR1 resulted in the abrogation of adiponectin-induced AMPK activation, whereas that of AdipoR2 resulted in decreased activity of PPAR-alpha signaling pathways. Simultaneous disruption of both AdipoR1 and R2 abolished adiponectin binding and actions, resulting in increased tissue triglyceride content, inflammation and oxidative stress, and thus leading to insulin resistance and marked glucose intolerance. Therefore, AdipoR1 and R2 serve as the predominant receptors for adiponectin in vivo and play important roles in the regulation of glucose and lipid metabolism, inflammation and oxidative stress in vivo.     

脂联素的研究进展

脂联素（adiponectin）是一种由脂肪细胞特异性高分泌,具有多种生物学功能的特殊蛋白质它直接作用于肝脏、骨骼肌和血管,能提高胰岛素敏感性,增强脂肪酸β氧化,抵制血管炎症反应,最新研究还发现脂联素和骨生成密切相关。与其它脂肪因子不同的是,循环中脂联素的浓度与人体脂肪含量成反比,会因TNF-α的作用而上调,会被噻唑烷二酮类药物所抑制,还受到胰岛素抵抗和炎症反应的影响脂联素受体有2类,分别为AdipoR1和AdipoR2,AdipoR1主要分布在骨骼肌上,AdipoR2则高表达于肝脏组织。本文主要综述了脂联素及其受体的结构、生物学功能和研究进展。     

The adiponectin receptor homologs in C. elegans promote energy utilization and homeostasis

Adiponectin is an adipokine with insulin-sensitising actions in vertebrates. Its receptors, AdipoR1 and AdipoR2, are PAQR-type proteins with 7-transmembrane domains and topologies reversed that of GPCR's, i.e. their C-termini are extracellular. We identified three adiponectin receptor homologs in the nematode C. elegans, named paqr-1, paqr-2 and paqr-3. These are differently expressed in the intestine (the main fat-storing tissue), hypodermis, muscles, neurons and secretory tissues, from which they could exert systemic effects. Analysis of mutants revealed that paqr-1 and -2 are novel metabolic regulators in C. elegans and that they act redundantly but independently from paqr-3. paqr-2 is the most important of the three paqr genes: mutants grow poorly, fail to adapt to growth at low temperature, and have a very high fat content with an abnormal enrichment in long (C20) poly-unsaturated fatty acids when combined with the paqr-1 mutation. paqr-2 mutants are also synthetic lethal with mutations in nhr-49, sbp-1 and fat-6, which are C. elegans homologs of nuclear hormone receptors, SREBP and FAT-6 (a Δ9 desaturase), respectively. Like paqr-2, paqr-1 is also synthetic lethal with sbp-1. Mutations in aak-2, the C. elegans homolog of AMPK, or nhr-80, another nuclear hormone receptor gene, suppress the growth phenotype of paqr-2 mutants, probably because they restore the balance between energy expenditure and storage. We conclude that paqr-1 and paqr-2 are receptors that regulate fatty acid metabolism and cold adaptation in C. elegans, that their main function is to promote energy utilization rather than storage, and that PAQR class proteins have regulated metabolism in metazoans for at least 700 million years.     

Detergent-free solubilisation & purification of a G protein coupled receptor using a polymethacrylate polymer

G protein coupled receptors (GPCRs) function as guanine nucleotide exchange factors (GEFs) at heterotrimeric G proteins, and conduct this role embedded in a lipid bilayer. Detergents are widely used to solubilise GPCRs for structural and biophysical analysis, but are poor mimics of the lipid bilayer and may be deleterious to protein function. Amphipathic polymers have emerged as promising alternatives to detergents, which maintain a lipid environment around a membrane protein during purification. Of these polymers, the polymethacrylate (PMA) polymers have potential advantages over the most popular styrene maleic acid (SMA) polymer, but to date have not been applied to purification of membrane proteins. Here we use a class A GPCR, neurotensin receptor 1 (NTSR1), to explore detergent-free purification using PMA. By using an NTSR1-eGFP fusion protein expressed in Sf9 cells, a range of solubilisation conditions were screened, demonstrating the importance of solubilisation temperature, pH, NaCl concentration and the relative amounts of polymer and membrane sample. PMA-solubilised NTSR1 displayed compatibility with standard purification protocols and millimolar divalent cation concentrations. Moreover, the receptor in PMA discs showed stimulation of both G_q and G_i1 heterotrimers to an extent that was greater than that for the detergent-solubilised receptor. PMA therefore represents a viable alternative to SMA for membrane protein purification and has a potentially broad utility in studying GPCRs and other membrane proteins.     

When worlds collide: IP3 receptors and the ERAD pathway

While cell signaling devotees tend to think of the endoplasmic reticulum (ER) as a Ca²⁺ store, those who study protein synthesis tend to see it more as site for protein maturation, or even degradation when proteins do not fold properly. These two worldviews collide when inositol 1,4,5-trisphosphate (IP₃) receptors are activated, since in addition to acting as release channels for stored ER Ca²⁺, IP₃ receptors are rapidly destroyed via the ER-associated degradation (ERAD) pathway, a ubiquitination- and proteasome-dependent mechanism that clears the ER of aberrant proteins. Here we review recent studies showing that activated IP₃ receptors are ubiquitinated in an unexpectedly complex manner, and that a novel complex composed of the ER membrane proteins SPFH1 and SPFH2 (erlin 1 and 2) binds to IP₃ receptors immediately after they are activated and mediates their ERAD. Remarkably, it seems that the conformational changes that underpin channel opening make IP₃ receptors resemble aberrant proteins, which triggers their binding to the SPFH1/2 complex, their ubiquitination and extraction from the ER membrane and finally, their degradation by the proteasome. This degradation of activated IP₃ receptors by the ERAD pathway serves to reduce the sensitivity of ER Ca²⁺ stores to IP₃ and may protect cells against deleterious effects of over-activation of Ca²⁺ signaling pathways.