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Agonist-activated glucagon receptors are deubiquitinated at early endosomes by two distinct deubiquitinases to facilitate Rab4a-dependent recycling.

Publication ,  Journal Article
Kaur, S; Chen, Y; Shenoy, SK
Published in: J Biol Chem
December 4, 2020

The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a seven-transmembrane G protein-coupled receptor (GPCR) that regulates blood glucose levels. Ubiquitination influences trafficking and signaling of many GPCRs, but its characterization for the GCGR is lacking. Using endocytic colocalization and ubiquitination assays, we have identified a correlation between the ubiquitination profile and recycling of the GCGR. Our experiments revealed that GCGRs are constitutively ubiquitinated at the cell surface. Glucagon stimulation not only promoted GCGR endocytic trafficking through Rab5a early endosomes and Rab4a recycling endosomes, but also induced rapid deubiquitination of GCGRs. Inhibiting GCGR internalization or disrupting endocytic trafficking prevented agonist-induced deubiquitination of the GCGR. Furthermore, a Rab4a dominant negative (DN) that blocks trafficking at recycling endosomes enabled GCGR deubiquitination, whereas a Rab5a DN that blocks trafficking at early endosomes eliminated agonist-induced GCGR deubiquitination. By down-regulating candidate deubiquitinases that are either linked with GPCR trafficking or localized on endosomes, we identified signal-transducing adaptor molecule-binding protein (STAMBP) and ubiquitin-specific protease 33 (USP33) as cognate deubiquitinases for the GCGR. Our data suggest that USP33 constitutively deubiquitinates the GCGR, whereas both STAMBP and USP33 deubiquitinate agonist-activated GCGRs at early endosomes. A mutant GCGR with all five intracellular lysines altered to arginines remains deubiquitinated and shows augmented trafficking to Rab4a recycling endosomes compared with the WT, thus affirming the role of deubiquitination in GCGR recycling. We conclude that the GCGRs are rapidly deubiquitinated after agonist-activation to facilitate Rab4a-dependent recycling and that USP33 and STAMBP activities are critical for the endocytic recycling of the GCGR.

Duke Scholars

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Published In

J Biol Chem

DOI

EISSN

1083-351X

Publication Date

December 4, 2020

Volume

295

Issue

49

Start / End Page

16630 / 16642

Location

United States

Related Subject Headings

  • rab5 GTP-Binding Proteins
  • rab4 GTP-Binding Proteins
  • Ubiquitination
  • Ubiquitin Thiolesterase
  • Receptors, Glucagon
  • RNA, Small Interfering
  • RNA Interference
  • Mutagenesis
  • Monensin
  • Humans
 

Citation

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Kaur, S., Chen, Y., & Shenoy, S. K. (2020). Agonist-activated glucagon receptors are deubiquitinated at early endosomes by two distinct deubiquitinases to facilitate Rab4a-dependent recycling. J Biol Chem, 295(49), 16630–16642. https://doi.org/10.1074/jbc.RA120.014532
Kaur, Suneet, Yuqing Chen, and Sudha K. Shenoy. “Agonist-activated glucagon receptors are deubiquitinated at early endosomes by two distinct deubiquitinases to facilitate Rab4a-dependent recycling.J Biol Chem 295, no. 49 (December 4, 2020): 16630–42. https://doi.org/10.1074/jbc.RA120.014532.
Kaur, Suneet, et al. “Agonist-activated glucagon receptors are deubiquitinated at early endosomes by two distinct deubiquitinases to facilitate Rab4a-dependent recycling.J Biol Chem, vol. 295, no. 49, Dec. 2020, pp. 16630–42. Pubmed, doi:10.1074/jbc.RA120.014532.

Published In

J Biol Chem

DOI

EISSN

1083-351X

Publication Date

December 4, 2020

Volume

295

Issue

49

Start / End Page

16630 / 16642

Location

United States

Related Subject Headings

  • rab5 GTP-Binding Proteins
  • rab4 GTP-Binding Proteins
  • Ubiquitination
  • Ubiquitin Thiolesterase
  • Receptors, Glucagon
  • RNA, Small Interfering
  • RNA Interference
  • Mutagenesis
  • Monensin
  • Humans