APPL Proteins FRET at the BAR: Direct Observation of APPL1 and APPL2 BAR Domain-Mediated Interactions on Cell Membranes Using FRET Microscopy

Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR) domain, a central pleckstrin homology (PH) domain, and a C-terminal phosphotyrosine binding (PTB) domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported.Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET) experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP) FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species.All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2) and heterotypic (i.e., APPL1-APPL2) manner on curved cell membranes. Furthermore, the results of our experiments did not show photoconversion of YFP into a CFP-like species following photobleaching, supporting the use of CFP donor/YFP acceptor FRET pairs in acceptor photobleaching studies

Genetic variations in APPL2 are associated with overweight and obesity in a Chinese population with normal glucose tolerance

<p>Abstract</p> <p>Background</p> <p>APPL1 and APPL2 are two adaptor proteins, which can mediate adiponectin signaling via binding to N terminus of adiponectin receptors in muscle cells. Genes encoding adiponectin and adiponectin receptors contribute to insulin resistance and the risk of obesity, and genetic variants of <it>APPL1 </it>are associated with body fat distribution. However, the association between genetic variations of <it>APPL2 </it>and metabolic traits remains unknown. In the current study, we aimed to test the impacts of <it>APPL2 </it>genetic variants on obesity in a Chinese population with normal glucose tolerance.</p> <p>Methods</p> <p>We genotyped six single nucleotide polymorphisms (SNPs) in <it>APPL2 </it>in 1,808 non-diabetic subjects. Overweight and obesity were defined by body mass index (BMI). Obesity-related anthropometric parameters were measured, including height, weight, waist circumference, hip circumference. BMI and waist-hip ratio (WHR) were calculated.</p> <p>Results</p> <p>We found significant evidence of association with overweight/obesity for rs2272495 and rs1107756. rs2272495 C allele and rs1107756 T allele both conferred a higher risk of being overweight and obese (OR 1.218, 95% CI 1.047-1.416, <it>p </it>= 0.011 for rs2272495; OR 1.166, 95% CI 1.014-1.341, <it>p </it>= 0.031 for rs1107756). After adjusting multiple comparisons, only the effect of rs2272495 on overweight/obesity remained to be significant (empirical <it>p </it>= 0.043). Moreover, we investigated the effects of these SNPs on obesity-related quantitative traits in all participants. rs2272495 was associated with BMI (<it>p </it>= 0.015), waist circumference (<it>p </it>= 0.006), hip circumference (<it>p </it>= 0.025) as well as WHR (<it>p </it>= 0.047) under a recessive model. Similar associations were found for rs1107756 except for WHR.</p> <p>Conclusion</p> <p>This study suggests that genetic variations in <it>APPL2 </it>are associated with overweight and obesity in Chinese population with normal glucose tolerance.</p

Structure of the APPL1 BAR-PH domain and characterization of its interaction with Rab5

APPL1 is an effector of the small GTPase Rab5. Together, they mediate a signal transduction pathway initiated by ligand binding to cell surface receptors. Interaction with Rab5 is confined to the amino (N)-terminal region of APPL1. We report the crystal structures of human APPL1 N-terminal BAR-PH domain motif. The BAR and PH domains, together with a novel linker helix, form an integrated, crescent-shaped, symmetrical dimer. This BAR–PH interaction is likely conserved in the class of BAR-PH containing proteins. Biochemical analyses indicate two independent Rab-binding sites located at the opposite ends of the dimer, where the PH domain directly interacts with Rab5 and Rab21. Besides structurally supporting the PH domain, the BAR domain also contributes to Rab binding through a small surface region in the vicinity of the PH domain. In stark contrast to the helix-dominated, Rab-binding domains previously reported, APPL1 PH domain employs β-strands to interact with Rab5. On the Rab5 side, both switch regions are involved in the interaction. Thus we identified a new binding mode between PH domains and small GTPases

Thyrotropin receptor trafficking relies on the hScrib–βPIX–GIT1–ARF6 pathway

G protein-coupled receptors are regulated by ligand stimulation, endocytosis, degradation of recycling to the cell surface. Little information is available on the molecular mechanisms underlying G protein-coupled receptors recycling. We have investigated recycling of the G protein-coupled thyroid stimulating hormone receptor (TSHR) and found that it relies on hScrib, a membrane-associated PDZ protein. hScrib directly binds to TSHR, inhibits basal receptor endocytosis and promotes recycling, and thus TSHR signalling, at the cell membrane. We previously demonstrated that hScrib is associated with a βPIX–GIT1 complex comprised of a guanine nucleotide exchange factor and a GTPase-activating protein for ADP ribosylation factors that is involved in vesicle trafficking. We used dominant-negative constructs and small interfering RNA to show that TSHR recycling is regulated by the interaction between hScrib and βPIX, and by the activity of GIT1. In addition, ARF6, a major target for GIT1, is activated during TSH stimulation of HEK293 and FRTL-5 thyroid cells, and plays a key role in TSHR recycling. Thus, we have uncovered an hScrib–βPIX–GIT1–ARF6 pathway devoted to TSHR trafficking and function