Thomas R F, Holt B D, Schwinn D A, Liggett S B
Department of Medicine (Pulmonary), Duke University Medical Center, Durham, NC 27710.
Proc Natl Acad Sci U S A. 1992 May 15;89(10):4490-4. doi: 10.1073/pnas.89.10.4490.
During continuous stimulation by agonist, beta 1- and beta 2-adrenergic receptors (ARs) undergo processes that lead to decreases in receptor expression. This receptor down-regulation serves to limit the cellular cAMP response during chronic agonist exposure. In the recently described third subtype of the beta AR, denoted beta 3AR, we found four potential cAMP response elements in the 5' flanking region, suggesting that expression of this receptor might be positively regulated by agonists. These elements were cloned into the vector pA10CAT2, which contains a chloramphenicol acetyltransferase reporter gene, and transiently expressed in VERO cells. Three of these elements, TGACTCCA, TGAGGTCT, and CGAGGTCA (located 518, 622, and 1125 bases upstream of the beta 3AR coding block, respectively) were found to increase transcription of the chloramphenicol acetyltransferase gene in response to cAMP analogues and agents that increase intracellular cAMP. 3T3-F442A cells, when differentiated into the adipocyte phenotype by insulin, expressed beta 3AR, and nuclear runoff studies from such cells confirmed cAMP enhancement of beta 3AR mRNA transcription. In these cells, beta 3AR mRNA increased in response to exposure to the beta 3AR agonist isoproterenol and remained elevated during exposures of up to 24-30 hr. During prolonged exposure to agonist, no downregulation of beta 3AR expression in 3T3-F442A cells occurred. Indeed, beta 3AR expression increased during agonist exposure to approximately 165% of basal expression. In marked contrast, beta 1AR expression declined by approximately 70% in response to chronic agonist exposure. These studies reveal a subtype-specific prolonged transcriptional regulation of a beta AR gene by the end product of its signal transduction pathway. Thus, the beta 3AR undergoes a paradoxical increase in receptor expression during chronic agonist exposure.
在激动剂持续刺激下，β1 - 和β2 - 肾上腺素能受体（ARs）会经历导致受体表达下降的过程。这种受体下调作用是为了在慢性激动剂暴露期间限制细胞内cAMP反应。在最近描述的β - AR的第三种亚型（称为β3AR）中，我们在其5'侧翼区域发现了四个潜在的cAMP反应元件，这表明该受体的表达可能受到激动剂的正向调节。这些元件被克隆到含有氯霉素乙酰转移酶报告基因的载体pA10CAT2中，并在VERO细胞中瞬时表达。发现其中三个元件，即TGACTCCA、TGAGGTCT和CGAGGTCA（分别位于β3AR编码区上游518、622和1125个碱基处），可响应cAMP类似物和增加细胞内cAMP的试剂而增加氯霉素乙酰转移酶基因的转录。3T3 - F442A细胞在胰岛素作用下分化为脂肪细胞表型时表达β3AR，对此类细胞进行的核转录分析证实了cAMP对β3AR mRNA转录的增强作用。在这些细胞中，β3AR mRNA在暴露于β3AR激动剂异丙肾上腺素后增加，并在长达24 - 30小时的暴露期间保持升高。在长时间暴露于激动剂期间，3T3 - F442A细胞中β3AR的表达没有下调。实际上，在激动剂暴露期间，β3AR的表达增加至基础表达的约165%。与之形成鲜明对比的是，β1AR的表达在慢性激动剂暴露后下降了约70%。这些研究揭示了β - AR基因的信号转导途径的终产物对其进行的亚型特异性长期转录调节。因此，β3AR在慢性激动剂暴露期间受体表达出现了反常的增加。
