The potent BECN2-ATG14 coiled-coil interaction is selectively critical for endolysosomal degradation of GPRASP1/GASP1-associated GPCRs

ABSTRACT

BECN2 is a mammal-specific homolog of BECN1. Both proteins serve as a scaffolding molecule in the class III phosphatidylinositol 3-kinase complex (PtdIns3K) to promote macroautophagy/autophagy and endolysosomal trafficking. Our previous studies have shown that the BECN1 coiled-coil domain forms a metastable homodimer and readily self-dissociate to form the BECN1-ATG14 or BECN1-UVRAG coiled-coil complex as part of the scaffolding “arm” of the PtdIns3K complex. Here we report the crystal structure of the BECN2 coiled-coil domain, which forms a metastable homodimer similar to BECN1 but shows reduced stability and large deviation from the ideal coiled-coil geometry due to extra “imperfect” residues. We also report the crystal structure of BECN2-ATG14 coiled-coil complex, which is structurally similar to the BECN1-UVRAG coiled-coil complex we reported previously but functionally distinct. The potent BECN2-ATG14 interaction is selectively critical for endolysosomal degradation of the GPRASP1-associated DRD2/D2R but shows limited effect on EGFR, a cargo that depends on the BECN1-UVRAG interaction. We designed stapled peptides that selectively interacted with the BECN2 coiled-coil domain to enhance the BECN2-ATG14 or BECN1-UVRAG interaction. One such peptide specifically promoted BECN2-dependent processes including autophagy and endolysosomal degradation of DRD2/D2R but did not affect BECN1-dependent EGFR degradation. Our findings suggest that, despite high sequence identity to BECN1, BECN2 coiled-coil domain has unique structural features and the BECN2-ATG14 interaction selectively promotes autophagic or endolysosomal degradation of BECN2-specific cargos including GPRASP1-associated GPCRs.

Abbreviations

AMBRA1 autophagy and beclin 1 regulator 1; ATG14 autophagy related 14; ATG5 autophagy related 5; ATG7 autophagy related 7; BECN1 beclin 1; BECN2 beclin 2; CC coiled-coil; CQ chloroquine CNR1/CB1R cannabinoid receptor 1 DAPI 4ʹ,6-diamidino-2-phenylindole; dCCD delete CCD; DRD2/D2R dopamine receptor D2 GPRASP1/GASP1 G protein-coupled receptor associated sorting protein 1 GPCR G-protein coupled receptor; ITC isothermal titration calorimetry; IP immunoprecipitation; KD knockdown; KO knockout; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; NRBF2 nuclear receptor binding factor 2; OPRD1/DOR opioid receptor delta 1 PIK3C3/VPS34 phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15 phosphoinositide-3-kinase regulatory subunit 4; PtdIns3K class III phosphatidylinositol 3-kinase; PtdIns3P phosphatidylinositol-3-phosphate; RUBCN rubicon autophagy regulator; SQSTM1/p62 sequestosome 1; UVRAG UV radiation resistance associated; VPS vacuolar protein sorting; WT wild type.

KEYWORDS:

• ATG14
• autophagy
• BECN2
• coiled-coil
• PtdIns3K

Acknowledgements

The work was supported by grants from the Research Grants Council of Hong Kong (PolyU 151052/16 M, PolyU 151015/17 M, PolyU 151062/18 M, 15103819, 15106421, R5050-18, and AoE/M-09/12), the Innovation and Technology Fund of Hong Kong (MRP/043/21), Shenzhen Basic Research Program of China (JCYJ20170818104619974, JCYJ20180306173813203, JCYJ20210324133803009) and Hong Kong Polytechnic University to YZ; Grants from the National Natural Science Foundation of China (Grant No. 81725022, 82173739, 81430083, 21661162003, 21472227), the Ministry of Science and Technology of China (National Key Research Program, Grant No. 2016YFA0502302), and Science and Technology Commission of Shanghai Municipality (Grant No. 20S11900500) to RW; Grants from NIH (Grant No. NIH R01 DK113170, R01 DK123447, R01 DA056720) and BrightFocus Foundation Alzheimer’s Disease Research Award to CH. Grants from the National Natural Science Foundation of China (32000864), Guangdong Basic and Applied Basic Research Foundation (2021A1515012054), and Discipline construction project of Guangdong Medical University to XQ.

All authors have given approval to the final version of the manuscript. The authors declare no competing financial interests. We thank Shanghai Synchrotron Radiation Facility (SSRF) beam line BL17U for help with data collection.

Disclosure statement

The authors have filed patent applications in U.S. and China based on this work.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2023.2233872

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.