Two distinct pathways for cAMP-mediated down-regulation of the beta 2-adrenergic receptor. Phosphorylation of the receptor and regulation of its mRNA level.
We have studied cyclic AMP-mediated regulation of the beta 2-adrenergic receptor (beta 2AR). The effects of cAMP were assessed in Chinese hamster fibroblast (CHW) cells expressing either the wild type human beta 2AR receptor (CH-beta 2) or mutated forms of the receptor lacking the consensus sequences for phosphorylation by the cAMP-dependent protein kinase. Treatment of the CH-beta 2 cells with the cAMP analogue dibutyryl cAMP (Bt2cAMP) induces a time-dependent "down-regulation" of the number of beta 2AR. This down-regulation of the receptors is accompanied by a decline in the steady state level of beta 2AR mRNA. Moreover, the treatment with Bt2cAMP induces an increase in the phosphorylation level of the membrane-associated beta 2AR. Both the reduction in beta 2AR mRNA and the enhanced phosphorylation of the receptor are rapid and precede the loss of receptor. The down-regulation of beta 2AR induced by Bt2cAMP is concentration-dependent and mimicked by the other biologically active cyclic nucleotide analogue, 8-Br-cAMP, by forskolin, and by the phosphodiesterase inhibitor, isobutylmethylxanthine. In the CHW cell lines expressing receptors lacking the putative protein kinase A phosphorylation sites, the Bt2cAMP-induced phosphorylation of beta 2AR is completely abolished. In these cells the down-regulation of beta 2AR receptor number produced by cAMP is significantly slowed, whereas the reduction in beta 2AR mRNA level is equivalent to that observed in CH-beta 2 cells. These data indicate that there are at least two pathways by which cAMP may decrease the number of beta 2ARs in cells: one involves phosphorylation of the receptor by the cAMP-dependent protein kinase and the other leads to a reduction in steady state beta 2AR mRNA levels.
Bouvier
M
M
Department of Medicine, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710.
Collins
S
S
O'Dowd
B F
BF
Campbell
P T
PT
de Blasi
A
A
Kobilka
B K
BK
MacGregor
C
C
Irons
G P
GP
Caron
M G
MG
Lefkowitz
R J
RJ
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D012333:RNA, Messenger; D011943:Receptors, Adrenergic, beta; D005576:Colforsin; D003994:Bucladesine; D004247:DNA; D013806:Theophylline; D000242:Cyclic AMP; D007545:Isoproterenol; D015056:1-Methyl-3-isobutylxanthine