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.

Ideally, therapy for autoimmune diseases should eliminate pathogenic autoimmune cells while sparing protective immunity, but feasible strategies for such an approach have been elusive. Here, we show that in the antibody-mediated autoimmune disease pemphigus vulgaris (PV), autoantigen-based chimeric immunoreceptors can direct T cells to kill autoreactive B lymphocytes through the specificity of the B cell receptor (BCR). We engineered human T cells to express a chimeric autoantibody receptor (CAAR), consisting of the PV autoantigen, desmoglein (Dsg) 3, fused to CD137-CD3ζ signaling domains. Dsg3 CAAR-T cells exhibit specific cytotoxicity against cells expressing anti-Dsg3 BCRs in vitro and expand, persist, and specifically eliminate Dsg3-specific B cells in vivo. CAAR-T cells may provide an effective and universal strategy for specific targeting of autoreactive B cells in antibody-mediated autoimmune disease.

2؉release from the endoplasmic reticulum, which plays a minor role in AMPK activation. Our results show that in muscle cells adiponectin is able to activate AMPK via two distinct mechanisms as follows: a major pathway (the APPL1/LKB1-dependent pathway) that promotes the cytosolic localization of LKB1 and a minor pathway (the phospholipase C/Ca 2؉ / Ca 2؉ /calmodulin-dependent protein kinase kinase-dependent pathway) that stimulates Ca 2؉ release from intracellular stores.Adiponectin, an adipokine abundantly expressed in adipose tissue, exhibits anti-diabetic, anti-inflammatory, and antiatherogenic properties and hence is a potential therapeutic target for various metabolic diseases (1-3). The beneficial effects of adiponectin are mediated through the direct interaction of adiponectin with its cell surface receptors, AdipoR1 and AdipoR2 (4, 5). Adiponectin increases fatty acid oxidation and glucose uptake in muscle cells by activating AMP-activated protein kinase (AMPK) 3 (4, 6), which depends on the interaction of AdipoR1 with the adaptor protein APPL1 (Adaptor protein containing Pleckstrin homology domain, Phosphotyrosine binding domain, and Leucine zipper motif) (5). However, the underlying mechanisms by which APPL1 mediates adiponectin signaling to AMPK activation and other downstream targets remain unclear.AMPK is a serine/threonine protein kinase that acts as a master sensor of cellular energy balance in mammalian cells by regulating glucose and lipid metabolism (7,8). AMPK is composed of a catalytic ␣ subunit and two noncatalytic regulatory subunits, ␤ and ␥. The NH 2 -terminal catalytic domain of the AMPK␣ subunit is highly conserved and contains the activating phosphorylation site (Thr 172 ) (9). Two AMPK variants, ␣1 and ␣2, exist in mammalian cells that show different localization patterns. AMPK␣1 subunit is localized in non-nuclear fractions, whereas the AMPK␣2 subunit is found in both nucleus and non-nuclear fractions (10). Biochemical regulation of AMPK activation occurs through various mechanisms. An increase in AMP level stimulates the binding of AMP to the ␥ subunit, which induces a conformational change in the AMPK heterotrimer and results in AMPK activation (11). Studies have shown that the increase in AMPK activity is not solely via AMPdependent conformational change, rather via phosphorylation by upstream kinases, LKB1 and CaMKK. Dephosphorylation by protein phosphatases is also important in regulating the activity of AMPK (12).LKB1 has been considered as a constitutively active serine/ threonine protein kinase that is ubiquitously expressed in all tissues (13,14). Under conditions of high cellular energy stress, LKB1 acts as the primary AMPK kinase through an AMP-dependent mechanism (15-17). Under normal physiological conditions, LKB1 is predominantly localized in the nucleus. LKB1 is translocated to the cytosol, either by forming a heterotrimeric complex with Ste20-related adaptor protein

APPL1 is a newly identified adiponectin receptor-binding protein that positively mediates adiponectin signaling in cells.Here we report that APPL2, an isoform of APPL1 that forms a dimer with APPL1, can interacts with both AdipoR1 and AdipoR2 and acts as a negative regulator of adiponectin signaling in muscle cells. Overexpression of APPL2 inhibits the interaction between APPL1 and AdipoR1, leading to down-regulation of adiponectin signaling in C2C12 myotubes. In contrast, suppressing APPL2 expression by RNAi significantly enhances adiponectin-stimulated glucose uptake and fatty acid oxidation. In addition to targeting directly to and competing with APPL1 in binding with the adiponectin receptors, APPL2 also suppresses adiponectin and insulin signaling by sequestrating APPL1 from these two pathways. In addition to adiponectin, metformin also induces APPL1-APPL2 dissociation. Taken together, our results reveal that APPL isoforms function as an integrated YinYang regulator of adiponectin signaling and mediate the crosstalk between adiponectin and insulin signaling pathways in muscle cells.Adiponectin, an adipocyte-secreted hormone that regulates energy homeostasis and insulin sensitivity, has been shown to be a promising therapeutic drug target for the treatment of type 2 diabetes (1-3). Adiponectin binds to its membrane receptors (AdipoR1 and AdipoR2) 3 and regulates lipid and glucose metabolism by activating downstream signaling molecules, such as AMP-activated protein kinase (AMPK), p38 MAP kinase (MAPK), and PPAR␣, in the muscle and liver (1, 4). Activation of AMPK by adiponectin reduces S6 kinase-mediated IRS-1 serine phosphorylation and increases IRS-1 tyrosine phosphorylation thus sensitizes insulin signaling in C2C12 myotubes (5), suggesting a direct cross-talk between the adiponectin and insulin signaling pathways.We have recently identified APPL1 (adaptor protein-containing PH domain, PTB domain, and leucine zipper motif) as a signaling protein immediately downstream of adiponectin receptors and positively mediates adiponectin signaling in muscle cells (6). This adaptor protein was previously shown to interact with the catalytic subunit of PI 3-kinase (p110) and Akt, which are two key kinases in the PI 3-kinase pathway downstream of the insulin receptor (7). The interaction between APPL1 and Akt is required for insulin-stimulated GLUT4 translocation (8) and for controlling Akt substrate selectivity (9). It has been shown that APPL1-potentiated Akt activity to suppress androgen receptor transactivation in prostate cancer cells (10). APPL1 has also been suggested to function as an adaptor protein in regulating follicle-stimulated hormone (FSH)-mediated PI 3-kinase/ Akt signaling pathway (11, 12). Our results showed that APPL1 binds directly to the intracellular part of the adiponectin receptors and positively mediates adiponectin signaling to the AMPK and p38 MAPK pathways, leading to increased glucose uptake and fatty acid oxidation in muscle cells (6). In addition, we found that APPL1 plays a critical ro...

Pemphigus vulgaris (PV) is an autoimmune epidermal blistering disease caused by autoantibodies directed against the desmosomal cadherin desmoglein-3 (Dsg3). Significant advances in our understanding of pemphigus pathomechanisms have been derived from the generation of pathogenic monoclonal Dsg3 antibodies. However, conflicting models for pemphigus pathogenicity have arisen from studies using either polyclonal PV patient IgG or monoclonal Dsg3 antibodies. In the present study, the pathogenic mechanisms of polyclonal PV IgG and monoclonal Dsg3 antibodies were directly compared. Polyclonal PV IgG cause extensive clustering and endocytosis of keratinocyte cell surface Dsg3, whereas pathogenic mouse monoclonal antibodies compromise cell-cell adhesion strength without causing these alterations in Dsg3 trafficking. Furthermore, tyrosine kinase or p38 MAPK inhibition prevents loss of keratinocyte adhesion in response to polyclonal PV IgG. In contrast, disruption of adhesion by pathogenic monoclonal antibodies is not prevented by these inhibitors either in vitro or in human skin explants. Our results reveal that the pathogenic activity of polyclonal PV IgG can be attributed to p38 MAPK-dependent clustering and endocytosis of Dsg3, whereas pathogenic monoclonal Dsg3 antibodies can function independently of this pathway. These findings have important implications for understanding pemphigus pathophysiology, and for the design of pemphigus model systems and therapeutic interventions.

Adiponectin functions as an insulin sensitizer, and yet the underlying molecular mechanism(s) remains largely unknown. We found that treating C2C12 myotubes with adiponectin or rapamycin enhanced the ability of insulin to stimulate IRS-1 tyrosine phosphorylation and Akt phosphorylation, concurrently with reduced p70 S6 kinase phosphorylation at Thr 389 as well as IRS-1 phosphorylation at Ser 302 and Ser 636/639 . Overexpression of dominant-negative AMP kinase (AMPK), but not dominant-negative p38 MAPK, reduced the insulin-sensitizing effect of adiponectin. Rapamycin, but not adiponectin, enhanced insulin-stimulated Akt phosphorylation in HeLa cells, which lack LKB1, and exogenous expression of LKB1 in HeLa cells rescued the insulin-sensitizing effect of adiponectin. Finally, overexpression of wild-type Rheb (Ras homology-enriched in brain) or the TSC2 mutant lacking the AMPK phosphorylation site (TSC2 S1345A ) inhibited the insulin-sensitizing effect of adiponectin in C2C12 cells. These results indicate that activation of the LKB1/AMPK/ TSC1/2 pathway alleviates the p70 S6 kinase-mediated negative regulation of insulin signaling, providing a mechanism by which adiponectin increases insulin sensitivity in cells.Adiponectin (Acrp30, AdipoQ, ApM1, and GBP28) is a collagen-like adipokine that has anti-atherogenic, antidiabetic, and insulin-sensitizing properties (1-3). Mice lacking adiponectin have severe hepatic insulin resistance (4), and administration of adiponectin to animal models of type 2 diabetes and insulin resistance significantly enhances insulin sensitivity (5). In humans, adiponectin levels are low in the plasma of obese and type 2 diabetes subjects (6 -8). The combined data suggest that adiponectin plays an important role in sensitizing insulin action.The molecular mechanism by which adiponectin acts as an insulin sensitizer remains largely unknown. Insulin initiates its action by binding to its membrane receptor, leading to tyrosine phosphorylation and activation of the insulin receptor (IR).2 A major pathway downstream of IR is the phosphatidylinositol 3-kinase (PI3K) signaling pathway, which mediates insulinstimulated GLUT4 membrane translocation and glucose uptake. Recent studies have shown that the PI3K pathway is negatively regulated by the TOR complex 1 (TORC1)-induced signaling pathway, which is mediated by the S6K-dependent phosphorylation of IRS-1 at several sites including Ser 302 , Ser 307 , and Ser 636/639 (9 -12). The inhibitory effect of S6K is greatly enhanced in cells lacking the tuberous sclerosis complex (TSC1/2), suggesting that TSC1/2 is a negative regulator of the mTOR/S6K signaling pathway (13). Interestingly, TSC1/2 activity is stimulated by AMP kinase (AMPK) (14), a heterotrimeric serine/threonine protein kinase, which is activated by adiponectin (15). However, whether activation of AMPK by adiponectin leads to inhibition of S6K and whether this inhibition plays a role in the insulin sensitizing effect of adiponectin remain unknown.In the present study, we have shown that adip...

Pemphigus vulgaris (PV) is a potentially fatal blistering disease characterized by autoantibodies against the desmosomal adhesion protein desmoglein (Dsg) 3. Whether autoantibody steric hindrance or signaling through pathways such as p38 MAPK is primary in disease pathogenesis is controversial. PV mAbs that cause endocytosis of Dsg3 but do not dissociate keratinocytes because of compensatory adhesion by Dsg1 do not activate p38. The same mAbs plus exfoliative toxin to inactivate Dsg1 but not exfoliative toxin alone activate p38, suggesting that p38 activation is secondary to loss of adhesion. Mice with epidermal p38␣ deficiency blister after passive transfer of PV mAbs; however, acantholytic cells retain cell surface Dsg3 compared with wild-type mice. In cultured keratinocytes, p38 knockdown prevents loss of desmosomal Dsg3 by PV mAbs, and exogenous p38 activation causes internalization of Dsg3, desmocollin 3, and desmoplakin. p38␣ MAPK is therefore not required for the loss of intercellular adhesion in PV, but may function downstream to augment blistering via Dsg3 endocytosis. Treatments aimed at increasing keratinocyte adhesion could be used in conjunction with immunosuppressive agents, potentially leading to safer and more effective combination therapy regimens. Pemphigus vulgaris (PV)2 is a potentially fatal autoimmune blistering disorder caused by autoantibodies to keratinocyte cell adhesion proteins known as desmogleins (1). The pathognomonic histologic finding in PV is suprabasal acantholysis, or the detachment of intact keratinocytes from each other because of loss of intercellular adhesion. A characteristic clinical finding in PV is Nikolsky's sign, in which blisters can be induced in otherwise normal-appearing skin by applying pressure or mechanical shear force, reflecting the loss of intercellular adhesion even in skin without overt blisters (2).PV autoantibodies primarily target desmoglein (Dsg) 3, a transmembrane adhesion molecule of desmosomes (3). Passive transfer experiments using neonatal mice have established the pathogenicity of the anti-Dsg3 autoantibodies in PV (4, 5). The anatomic site of blister formation is thought to be due to the tissue-specific expression patterns of different Dsg isoforms, also known as the desmoglein compensation theory. Dsg3 is expressed by basal keratinocytes of mucosa and skin, whereas Dsg1 is expressed by basal keratinocytes in skin but not mucosa (6, 7). Therefore, patients with Dsg3 autoantibodies demonstrate blistering in the mucosa, where compensatory adhesion by Dsg1 is not present (mucosal-dominant PV). In some patients who progress to develop Dsg1 in addition to Dsg3 autoantibodies, suprabasal blisters appear in both the mucosa and skin (mucocutaneous PV) (8 -10).Epitope mapping studies have shown that pathogenic PV autoantibodies preferentially bind the amino-terminal domain of Dsg3 that is predicted to form the transadhesive interface between cells, based on analogy to ultrastructural models for the classical cadherins (11-13). PV IgG has also been shown...

APPL1 interacts with adiponectin receptors and other important signaling molecules. It contains a BAR and a PH domain near its N terminus, and the two domains may function as a unit (BAR-PH domain). We report here the crystal structures of the BAR-PH and PTB domains of human APPL1. The structures reveal novel features for BAR domain dimerization and for the interactions between the BAR and PH domains. The BAR domain dimer of APPL1 contains two four-helical bundles, whereas other BAR domain dimers have only three helices in each bundle. The PH domain is located at the opposite ends of the BAR domain dimer. Yeast two-hybrid assays confirm the interactions between the BAR and PH domains. Lipid binding assays show that the BAR, PH, and PTB domains can bind phospholipids. The ability of APPL1 to interact with multiple signaling molecules and phospholipids supports an important role for this adaptor in cell signaling.