Abstract
Down-regulation is a classic response of most G protein-coupled receptors to prolonged agonist stimulation. We recently showed that when expressed in baby hamster kidney cells, the human beta1-but not the beta2-adrenergic receptor (AR) is totally resistant to agonist-mediated down-regulation, whereas both have similar rates of basal degradation (Liang, W., Austin, S., Hoang, Q., and Fishman, P. H. (2003) J. Biol. Chem. 278, 39773-39781). To identify the underlying mechanism(s) for this resistance, we investigated the role of proteasomes, lysosomes, and ubiquitination in the degradation of beta1AR expressed in baby hamster kidney and human embryonic kidney 293 cells. Both lysosomal and proteasomal inhibitors reduced beta1AR degradation in agonist-stimulated cells but were less effective on basal degradation. To determine whether beta1AR trafficked to lysosomes we used confocal fluorescence microscopy. We observed some colocalization of beta1AR and lysosomal markers in agonist-treated cells but much less than that of beta2AR even in cells co-transfected with arrestin-2, which increases beta1AR internalization. Ubiquitination of beta2AR readily occurred in agonist-stimulated cells, whereas ubiquitination of beta1AR was not detectable even under conditions optimal for that of beta2AR. Moreover, in cells expressing betaAR chimeras in which the C termini have been switched, the chimeric beta1AR with beta2AR C-tail underwent ubiquitination and down-regulation, but the chimeric beta2AR with beta1AR C-tail did not. Our results demonstrate for the first time that beta1AR and beta2AR differ in the ability to be ubiquitinated. Because ubiquitin serves as a signal for sorting membrane receptors to lysosomes, the lack of agonist-mediated ubiquitination of beta1AR may prevent its extensive trafficking to lysosomes and, thus, account for its resistance to down-regulation.
Highlights
From the Membrane Biochemistry Section, Laboratory of Molecular and Cellular Neurobiology, NINDS, National Institutes of Health, Bethesda, Maryland 20892
We recently showed that when expressed in baby hamster kidney cells, the human 1- but not the 2adrenergic receptor (AR) is totally resistant to agonistmediated down-regulation, whereas both have similar rates of basal degradation
To identify the underlying mechanism(s) for this resistance, we investigated the role of proteasomes, lysosomes, and ubiquitination in the degradation of 1AR expressed in baby hamster kidney and human embryonic kidney 293 cells
Summary
To identify the underlying mechanism(s) for this resistance, we investigated the role of proteasomes, lysosomes, and ubiquitination in the degradation of 1AR expressed in baby hamster kidney and human embryonic kidney 293 cells. Both lysosomal and proteasomal inhibitors reduced 1AR degradation in agonist-stimulated cells but were less effective on basal degradation. In one study proteasomal but not lysosomal inhibitors reduced the down-regulation of and ␦ opioid receptors [14], whereas in another, lactacystin, a very specific proteasomal inhibitor, did not impair agonist-induced internalization, lysosomal targeting, and degradation of the ␦ opioid receptor [15] Some of these studies found that steadystate receptor levels increase in unstimulated cells treated with. The inability of the C-tail of 1AR to facilitate receptor ubiquitination may be the basis for its insensitivity to agonistmediated degradation
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