Huntingtin-associated Protein 1 Interacts with Hepatocyte Growth Factor-regulated Tyrosine Kinase Substrate and Functions in Endosomal Trafficking

Abstract

Huntingtin-associated protein 1 (HAP1) is a novel protein of unknown function with a higher binding affinity for the mutant form of Huntington's disease protein huntingtin. Here we report that HAP1 interacts with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a mammalian homologue of yeast vacuolar protein sorting protein Vps27p involved in the endosome-to-lysosome trafficking. This novel interaction was identified in a yeast two-hybrid screen using fulllength Hrs as bait, and confirmed by in vitro binding assays and co-immunoprecipitation experiments. Deletion analysis reveals that the association of HAP1 with Hrs is mediated via a coiled-coil interaction between the central coiled-coil domains of both proteins. Immunofluorescence and subcellular fractionation studies show that HAP1 co-localizes with Hrs on early endosomes. Like Hrs, overexpression of HAP1 causes the formation of enlarged early endosomes, and inhibits the degradation of internalized epidermal growth factor receptors. Whereas overexpression of HAP1 does not affect either constitutive or ligand-induced receptor-mediated endocytosis, it potently blocks the trafficking of endocytosed epidermal growth factor receptors from early endosomes to late endosomes. These findings implicate, for the first time, the involvement of HAP1 in the regulation of vesicular trafficking from early endosomes to the late endocytic compartments.

Huntingtin-associated protein 1 regulates inhibitory synaptic transmission by modulating γ-aminobutyric acid type A receptor membrane trafficking

“…HAP1 exhibited clusters in the dendrites and axons, in addition to substantial intracellular somatic staining, including large perinuclear structures consistent with the localization of this protein to intracellular compartments such as endosomes and tubulovesicular structures (Fig. 2G), as demonstrated (23)(24)(25)(26). The GABA A R ␥2 subunit appeared as membrane clusters in addition to abundant intracellular aggregates (Fig.…”

“…Future studies using loss-of-function approaches should help clarify this issue. Previous studies have shown that HAP1 associates with proteins that participate in vesicular membrane trafficking, including p150 glued , a component of the dynactin complex, essential for the activity of the microtubule motor protein dynein and Hrs early endosomal phosphoprotein involved in the sorting of ubiquitinated membrane proteins (26,(30)(31)(32). Therefore it is tempting to speculate that HAP1 regulates GABA A R endocytic sorting by facilitating interactions with microtubule motors such as dynein or kinesin (30,31,33,34).…”

“…GABA A Rs undergo clathrin-mediated endocytosis and can be detected in endocytic structures (1,8,10,11,27). HAP1 has been localized to clathrin-coated vesicles and the endocytic pathway (23)(24)(25)(26) and has been implicated in endocytic protein trafficking (26), suggesting that HAP1 may be involved in the endocytic sorting of GABA A Rs. To further examine the effects of HAP1 on the endocytic sorting of GABA A Rs, cultured cortical neurons were nucleofected with a HAP1 cDNA to modify HAP1 expression levels.…”

Dendritic spine loss and neurodegeneration is rescued by Rab11 in models of Huntington's disease

“…22 Synaptic components are also retrieved from the plasma membrane by endocytosis into REs and can be either reinserted or degraded. 23 As vesicle trafficking events have been implicated in HD, [24][25][26][27][28][29] we decided to investigate whether aggregation of a mutant htt exon-1 fragment in the cytoplasm would affect spine maintenance by interfering with endosomal trafficking. Previous reports have shown a disruption of endocytic trafficking in cells expressing a mutant htt fragment, 28,29 which appears to be mediated by Rab11, a GTPase involved in RE function.…”

DYRK1A regulates Hap1–Dcaf7/WDR68 binding with implication for delayed growth in Down syndrome

“…Thus, we concluded that Hap1 not only sequesters but also stabilizes Dcaf7 in the cytoplasm by inhibiting its degradation by the UPS. Because Hap1 can protect internalized receptors from lysosomal degradation (11,(29)(30)(31), Hap1 may stabilize its binding partners from subsequent degradation through either the lysosome or UPS.…”