LETTERS 516 NATURE CELL BIOLOGY VOLUME 8 | NUMBER 5 | MAY 2006 APPL1 binds to adiponectin receptors and mediates adiponectin signalling and function Xuming Mao 1 , Chintan K. Kikani 2 , Ramon A. Riojas 2 , Paul Langlais 2 , Lixin Wang 1 , Fresnida J. Ramos 1 , Qichen Fang 1,5 , Christine Y. Christ-Roberts 1 , Jenny Y. Hong 3 , Ryang-Yeo Kim 1 , Feng Liu 1,2,4 and Lily Q. Dong 1,3,4,6 Adiponectin, also known as Acrp30, is an adipose tissue- derived hormone with anti-atherogenic, anti-diabetic and insulin sensitizing properties 1–3 . Two seven-transmembrane domain-containing proteins, AdipoR1 and AdipoR2, have recently been identified as adiponectin receptors 4 , 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. Adiponectin is an adipose tissue-derived hormone that has important functions in the regulation of lipid and glucose metabolism 1–3 . In mice, targeted deletion of the adiponectin gene leads to insulin resistance 5,6 . In addition, administration by continuous systemic infusion of adiponectin significantly increased insulin sensitivity in type 2 diabetic mice 7 . In humans, a reduced serum concentration of adiponectin has been shown to correlate with obesity 8 , insulin resistance 7,9 and type 2 diabetes 9–11 . Collectively, these findings suggest that adiponectin has an essential function in regulating whole-body energy homeostasis and indicate that this adipose hormone is a strong candidate for the development of drugs to treat insulin resistance and type 2 diabetes. The signalling mechanisms responsible for the action of adiponec- tin remain largely unknown. Two adiponectin receptors, AdipoR1 and AdipoR2, have recently been identified 4 . These receptors contain seven transmembrane domains and are structurally and functionally distinct from G-protein-coupled receptors (GPCR). Unlike GPCRs, the amino (N)-termini of AdipoR1 and AdipoR2 are intracellular 4 (see Supplementary Information, Fig. S1a). In agreement with this result, it was found that adiponectin interacts with the carboxyl (C)- terminus of AdipoR1 when the yeast two-hybrid system was used to identify adiponectin interacting proteins (see Supplementary Information, Fig. S1b). To identify proteins that interact with the adiponectin receptor, a yeast two-hybrid cDNA library derived from human fetal brain 12 was screened using the intracellular portion of mouse AdipoR1 (amino acids 4–142) as bait. Sequence analysis revealed that 7 of the 17 positive clones contained a cDNA encoding the C-terminus of an adaptor protein previously named APPL 13 or DIP13α 14 (deleted in colorectal cancer (DCC)-inter- acting protein; Fig. 1a). A putative APPL-related protein was identified in the GeneBank database (accession number BC048906 (mouse) and NM_018171 (human)) that shares a 54% identity in protein sequence to APPL, suggesting the presence of an isoform of this protein (APPL2). Western blot analysis revealed that APPL1 is highly expressed in dif- ferentiated C2C12 myotubes, INS1 insulinoma cells and L6 cells (Fig. 1b). Moderate expression of APPL1 was detected in HEK293, mouse hepa- tocytes and non-differentiated C2C12 myoblasts (Fig. 1b and data not shown). APPL1 expression was also observed in mouse brain, skeletal muscle, fat, heart, spleen and to a lesser extent, pancreas and kidney tis- sues (Fig. 1c). The findings that APPL1 is highly expressed in both differ- entiated C2C12 myotubes and mouse skeletal muscle suggest a potential role for this adaptor protein in regulating the insulin-sensitizing effect of adiponectin in skeletal muscle. In agreement with this, AdipoR1 was highly expressed in this insulin-sensitive tissue (Fig. 1c). 1 Departments of Pharmacology, 2 Biochemistry, 3 Cellular and Structural Biology, and 4 the Barshop Center for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio, TX 78229–3900, USA. 5 Permanent address: Shanghai Diabetes Institute, Shanghai Jiaotong University Affiliated Sixth Hospital, Shanghai 200233, People’s Republic of China. 6 Correspondence should be addressed to L.Q.D. (e-mail: dongq@uthscsa.edu) Received 8 November 2005; accepted 17 March 2006; published online 16 April 2006; corrected after print 1 June 2006; DOI: 10.1038/ncb1404 © 2006 Nature Publishing Group