Abstract
Prolonged agonist stimulation results in down-regulation of most G protein-coupled receptors. When we exposed baby hamster kidney cells stably expressing the human beta1-adrenergic receptor (beta 1AR) to agonist over a 24-h period, we instead observed an increase of approximately 30% in both beta 1AR binding activity and immune-detected receptors. In contrast, beta 2AR expressed in these cells exhibited a decrease of > or =50%. We determined that the basal turnover rates of the two subtypes were similar (t(1/2) approximately 7 h) and that agonist stimulation increased beta 2AR but not beta 1AR turnover. Blocking receptor trafficking to lysosomes with bafilomycin A1 had no effect on basal turnover of either subtype but blocked agonist-stimulated beta 2AR turnover. As beta 1AR mRNA levels increased in agonist-stimulated cells, beta 1AR up-regulation appeared to result from increased synthesis with no change in degradation. To explore the basis for the subtype differences, we expressed chimeras in which the C termini had been exchanged. Each chimera responded to persistent agonist stimulation based on the source of its C-tail; beta 1AR with a beta 2AR C-tail underwent down-regulation, and beta 2AR with a beta 1AR C-tail underwent up-regulation. The C-tails had a corresponding effect on agonist-stimulated receptor phosphorylation and internalization with the order being beta 2AR > beta 1AR with beta 2AR C-tail > beta 2AR with a beta 1AR C-tail > beta 1AR. As internalization may be a prerequisite for down-regulation, we addressed this possibility by co-expressing each subtype with arrestin-2. Although beta 1AR internalization was increased to that of beta 2AR, down-regulation still did not occur. Instead, beta 1AR accumulated inside the cells. We conclude that in unstimulated cells, both subtypes appear to be turned over by the same mechanism. Upon agonist stimulation, both subtypes are internalized, and beta 2AR but not beta 1AR undergoes lysosomal degradation, the fate of each subtype being regulated by determinants in its C-tail.
Highlights
From the Membrane Biochemistry Section, Laboratory of Molecular and Cellular Neurobiology, NINDS, National Institutes of Health, Bethesda, Maryland 20892
Prolonged 1AR Regulation Unaffected by Increased Internalization—1AR is more resistant to agonist-mediated internalization compared with 2AR in CHW [25, 26, 31] and HEK 293 cells [28, 32]2 As endocytosis of some GPCRs is necessary for their down-regulation [6], including 1AR [27] and 2AR [7, 8], we explored the possibility that subtype differences in internalization might account for differences in down-regulation in BHK cells
To resolve how the two processes contribute to this subtype difference, we determined both the levels of AR mRNA and the kinetics of AR degradation
Summary
Vol 278, No 41, Issue of October 10, pp. 39773–39781, 2003 Printed in U.S.A. Resistance of the Human 1-Adrenergic Receptor to Agonist-mediated Down-regulation. We conclude that in unstimulated cells, both subtypes appear to be turned over by the same mechanism Upon agonist stimulation, both subtypes are internalized, and 2AR but not 1AR undergoes lysosomal degradation, the fate of each subtype being regulated by determinants in its C-tail. We initiated the present studies to identify some of the mechanisms involved in the resistance to down-regulation of human 1AR during persistent agonist stimulation. Our results indicate that in BHK cells, 1AR is resistant to agonist-mediated down-regulation and instead undergoes up-regulation as does its mRNA; increased 1AR internalization does not result in down-regulation; basal turnover is similar for both subtypes and appears to be non-lysosomal whereas agonist-mediated turnover of 2AR is lysosomal; and the C-tails are key determinants of down-regulation, the 1AR C-tail conferring resistance and the 2AR conferring C-tail susceptibility
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