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Beta2-adrenergic receptor lysosomal trafficking is regulated by ubiquitination of lysyl residues in two distinct receptor domains.

Publication ,  Journal Article
Xiao, K; Shenoy, SK
Published in: J Biol Chem
April 8, 2011

Agonist stimulation of the β2-adrenergic receptors (β2ARs) leads to their ubiquitination and lysosomal degradation. Inhibition of lysosomal proteases results in the stabilization and retention of internalized full-length β2ARs in the lysosomes, whereas inhibition of proteasomal proteases stabilizes newly synthesized β2ARs in nonlysosomal compartments. Additionally, a lysine-less β2AR (0K-β2AR) that is deficient in ubiquitination and degradation is not sorted to lysosomes unlike the WT β2AR, which is sorted to lysosomes. Thus, lysosomes are the primary sites for the degradation of agonist-activated, ubiquitinated β2ARs. To identify the specific site(s) of ubiquitination required for lysosomal sorting of the β2AR, four mutants, with lysines only in one intracellular domain, namely, loop 1, loop 2, loop 3, and carboxyl tail were generated. All of these receptor mutants coupled to G proteins, recruited β-arrestin2, and internalized just as the WT β2AR. However, only loop 3 and carboxyl tail β2ARs with lysines in the third intracellular loop or in the carboxyl tail were ubiquitinated and sorted for lysosomal degradation. As a complementary approach, we performed MS-based proteomic analyses to directly identify ubiquitination sites within the β2AR. We overexpressed and purified the β2AR from HEK-293 cells with or without prior agonist exposure and subjected trypsin-cleaved β2AR to LC-MS/MS analyses. We identified ubiquitinated lysines in the third intracellular loop (Lys-263 and Lys-270) and in the carboxyl tail (Lys-348, Lys-372, and Lys-375) of the β2AR. These findings introduce a new concept that two distinct domains in the β2AR are involved in ubiquitination and lysosomal degradation, contrary to the generalization that such regulatory mechanisms occur mainly at the carboxyl tails of GPCRs and other transmembrane receptors.

Duke Scholars

Published In

J Biol Chem

DOI

EISSN

1083-351X

Publication Date

April 8, 2011

Volume

286

Issue

14

Start / End Page

12785 / 12795

Location

United States

Related Subject Headings

  • Ubiquitination
  • Tandem Mass Spectrometry
  • Receptors, Adrenergic, beta-2
  • Proteomics
  • Protein Transport
  • Proteasome Endopeptidase Complex
  • Microscopy, Confocal
  • Mass Spectrometry
  • Lysosomes
  • Isoproterenol
 

Citation

APA
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ICMJE
MLA
NLM
Xiao, K., & Shenoy, S. K. (2011). Beta2-adrenergic receptor lysosomal trafficking is regulated by ubiquitination of lysyl residues in two distinct receptor domains. J Biol Chem, 286(14), 12785–12795. https://doi.org/10.1074/jbc.M110.203091
Xiao, Kunhong, and Sudha K. Shenoy. “Beta2-adrenergic receptor lysosomal trafficking is regulated by ubiquitination of lysyl residues in two distinct receptor domains.J Biol Chem 286, no. 14 (April 8, 2011): 12785–95. https://doi.org/10.1074/jbc.M110.203091.
Xiao, Kunhong, and Sudha K. Shenoy. “Beta2-adrenergic receptor lysosomal trafficking is regulated by ubiquitination of lysyl residues in two distinct receptor domains.J Biol Chem, vol. 286, no. 14, Apr. 2011, pp. 12785–95. Pubmed, doi:10.1074/jbc.M110.203091.

Published In

J Biol Chem

DOI

EISSN

1083-351X

Publication Date

April 8, 2011

Volume

286

Issue

14

Start / End Page

12785 / 12795

Location

United States

Related Subject Headings

  • Ubiquitination
  • Tandem Mass Spectrometry
  • Receptors, Adrenergic, beta-2
  • Proteomics
  • Protein Transport
  • Proteasome Endopeptidase Complex
  • Microscopy, Confocal
  • Mass Spectrometry
  • Lysosomes
  • Isoproterenol