Bouvier M, Collins S, O'Dowd B F, Campbell P T, de Blasi A, Kobilka B K, MacGregor C, Irons G P, Caron M G, Lefkowitz R J
Department of Medicine, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 1989 Oct 5;264(28):16786-92.
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.
我们研究了环磷酸腺苷（cAMP）介导的β2-肾上腺素能受体（β2AR）的调节作用。在中国仓鼠成纤维细胞（CHW）中，评估了cAMP对表达野生型人β2AR受体（CH-β2）或缺乏依赖cAMP的蛋白激酶磷酸化共有序列的受体突变形式的影响。用cAMP类似物二丁酰cAMP（Bt2cAMP）处理CH-β2细胞会诱导β2AR数量随时间的“下调”。受体的这种下调伴随着β2AR mRNA稳态水平的下降。此外，用Bt2cAMP处理会导致膜相关β2AR的磷酸化水平增加。β2AR mRNA的减少和受体磷酸化的增强都是快速的，且先于受体的丢失。Bt2cAMP诱导的β2AR下调是浓度依赖性的，并且被另一种生物活性环核苷酸类似物8-溴-cAMP、福斯可林和磷酸二酯酶抑制剂异丁基甲基黄嘌呤模拟。在表达缺乏假定蛋白激酶A磷酸化位点的受体的CHW细胞系中，Bt2cAMP诱导的β2AR磷酸化完全被消除。在这些细胞中，cAMP产生的β2AR受体数量下调明显减慢，而β2AR mRNA水平的降低与在CH-β2细胞中观察到的相当。这些数据表明，cAMP可能通过至少两条途径减少细胞中β2AR的数量：一条途径涉及依赖cAMP的蛋白激酶对受体的磷酸化，另一条途径导致β2AR mRNA稳态水平的降低。
