Resistance of the human β1-adrenergic receptor to agonist-mediated down-regulation -: Role of the C terminus in determining β-subtype degradation

Prolonged agonist stimulation results in down-regulation of most G protein-coupled receptors. When we exposed baby hamster kidney cells stably expressing the human beta(1)-adrenergic receptor (beta(1)AR) to agonist over a 24-h period, we instead observed an increase of similar to30% in both beta(1)AR binding activity and immune-detected receptors. In contrast, beta(2)AR expressed in these cells exhibited a decrease of greater than or equal to50%. We determined that the basal turnover rates of the two subtypes were similar (t1/2 similar to 7 h) and that agonist stimulation increased beta(2)AR but not beta(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 beta(2)AR turnover. As beta(1)AR mRNA levels increased in agonist-stimulated cells, beta(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; beta(1)AR with a beta(2)AR C-tail underwent down-regulation, and beta(2)AR with a beta(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 beta(2)AR > beta(1)AR with beta(2)AR C-tail > beta(2)AR with a beta(1)AR C-tail > beta(1)AR. As internalization may be a prerequisite for down-regulation, we addressed this possibility by co-expressing each subtype with arrestin-2. Although beta(1)AR internalization was increased to that of beta(2)AR, down-regulation still did not occur. Instead, beta(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 beta(2)AR but not beta(1)AR undergoes lysosomal degradation, the fate of each subtype being regulated by determinants in its C-tail.