Accelerated dephosphorylation of the beta2-adrenergic receptor by mutation of the C-terminal lysines: effects on ubiquitination, intracellular trafficking, and degradation.
Agonist-mediated ubiquitination regulates some G protein-coupled receptors by targeting them to lysosomes for degradation. Phosphorylation also regulates receptor endocytosis and trafficking to lysosomes. To explore the roles of the two post-translational modifications, we mutated the three C-terminal lysines to arginines in the human beta 2-adrenergic receptor (beta 2AR) (K348/372/375R). The level of agonist-mediated ubiquitination of the mutant (3K/R) was greatly reduced compared to that of wild-type (WT) beta 2AR in whole cells and in cell-free assays. Downregulation of 3K/R also was attenuated compared to that of the WT, whereas internalization and recycling were more similar. During endocytosis, WT and 3K/R appeared in different vesicles and WT, but not 3K/R, was transported to lysosomes. Both were rapidly phosphorylated in agonist-stimulated cells, but upon agonist removal, the rate of dephosphorylation of 3K/R initially was approximately 5 times faster than that of WT. The increased rate also was observed in a cell-free, soluble assay and, thus, was not due to differences in receptor trafficking. Okadaic acid, a potent phosphatase inhibitor, reduced the level of dephosphorylation and increased the levels of lysosomal targeting and degradation of 3K/R. The reduced level of ubiquitination and rapid dephosphorylation of 3K/R appear to prevent it from being sorted to lysosomes in contrast to the phosphorylated and ubiquitinated WT beta 2AR. Our findings indicate that both phosphorylation and ubiquitination are involved in the intracellular sorting of beta 2AR between pathways of recycling to the plasma membrane and degradation in lysosomes, and that the rate of dephosphorylation may be another mechanism of regulating the sorting.
Liang
Wei
W
Membrane Biochemistry Section, Laboratory of Molecular and Cellular Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
Hoang
Quang
Q
Clark
Richard B
RB
Fishman
Peter H
PH
D002460:Cell Line; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D009154:Mutation; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D054875:Ubiquitination
D018343:Receptors, Adrenergic, beta-2; D008239:Lysine