Prolonged agonist stimulation results in down-regulation of most G protein-coupled receptors. When we exposed baby hamster kidney cells stably expressing the human  1 -adrenergic receptor ( 1 AR) to agonist over a 24-h period, we instead observed an increase of ϳ30% in both  1 AR binding activity and immune-detected receptors. In contrast,  2 AR expressed in these cells exhibited a decrease of >50%. We determined that the basal turnover rates of the two subtypes were similar (t1 ⁄2 ϳ 7 h) and that agonist stimulation increased  2 AR but not  1 AR turnover. Blocking receptor trafficking to lysosomes with bafilomycin A 1 had no effect on basal turnover of either subtype but blocked agonist-stimulated  2 AR turnover. As  1 AR mRNA levels increased in agonist-stimulated cells,  1 AR 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;  1 AR with a  2 AR C-tail underwent down-regulation, and  2 AR with a  1 AR C-tail underwent up-regulation. The C-tails had a corresponding effect on agonist-stimulated receptor phosphorylation and internalization with the order being  2 AR >  1 AR with  2 AR C-tail >  2 AR with a  1 AR C-tail >  1 AR. As internalization may be a prerequisite for down-regulation, we addressed this possibility by co-expressing each subtype with arrestin-2. Although  1 AR internalization was increased to that of  2 AR, down-regulation still did not occur. Instead,  1 AR 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  2 AR but not  1 AR undergoes lysosomal degradation, the fate of each subtype being regulated by determinants in its C-tail.
SummaryDifferences in endosomal targeting of human β β 1 -and β β 2 -adrenergic receptors following clathrin-mediated endocytosis
Prolonged agonist exposure often induces downregulation of G protein-coupled receptors (GPCRs). Although downregulation of the prototypical beta(2)-adrenergic receptor (beta(2)AR) has been extensively studied, the underlying mechanisms have yet to be resolved. As even less is known about the beta(1)-subtype, we investigated the downregulation of human beta(1)AR stably expressed in Chinese hamster fibroblasts in response to the agonist isoproterenol or the cell-permeable, chlorophenylthio-cAMP (CPT-cAMP). While either effector mediated decreases in both beta(1)AR binding activity and steady-state beta(1)AR mRNA levels, there were significant differences in their actions. Whereas agonist-mediated downregulation of beta(1)AR followed first-order kinetics, that induced by CPT-cAMP was delayed for several hours and approximately 50% of the former. Furthermore, agonist but not CPT-cAMP induced beta(1)AR internalization, and inhibiting internalization also suppressed agonist-mediated downregulation. The latter, however, was more sensitive than the former to agonist concentration (EC(50) of 0.3 vs 48 nM). Thus, at < or =1 nM agonist, downregulation occurred without internalization and with a pattern similar to that mediated by CPT-cAMP. The amounts of beta(1)AR downregulated or internalized were proportional to initial receptor levels but reached saturation at approximately 2 and 3 pmol/mg of protein, respectively. The fate of beta(1)AR protein during downregulation was determined by immunoblotting with anti-C-terminal antibodies. In agonist-treated cells, beta(1)AR protein disappeared with time and without any immunoreactive degradation products. Agonist-mediated downregulation of the human beta(1)AR appears to be a complex process that consists of both agonist- and cAMP-specific components. The former involves both receptor internalization and degradation whereas the latter involves a reduction in receptor mRNA.
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