Resistance of the human beta 1-adrenergic receptor to agonist-mediated down-regulation. Role of the C terminus in determining beta-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 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.
Liang
Wei
W
Membrane Biochemistry Section, Laboratory of Molecular and Cellular Neurobiology, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
Austin
Steven
S
Hoang
Quang
Q
Fishman
Peter H
PH
D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018342:Receptors, Adrenergic, beta-1 / Q000737:chemistry / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000318:Adrenergic beta-Agonists; D018076:DNA, Complementary; D012333:RNA, Messenger; D018342:Receptors, Adrenergic, beta-1; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins