Green S A, Liggett S B
Department of Medicine (Pulmonary), University of Cincinnati Medical Center, Ohio 45267-0564.
J Biol Chem. 1994 Oct 21;269(42):26215-9.
beta-Adrenergic receptor (beta AR) subtypes differ not only by characteristic ligand affinities but also in the manner and extent to which they mediate agonist-promoted events such as activation of adenylyl cyclase and receptor sequestration. We utilized mutagenesis and recombinant expression in Chinese hamster fibroblasts to examine the effect of an unusual proline-rich 24-amino acid sequence (PARPPSPSPSPVPAPAPPPGPPRP) present in the third intracellular loop of the beta 1AR, but not in the beta 2AR, on the aforementioned receptor-mediated events. Cells expressing the wild-type beta 2AR stimulated adenylyl cyclase in response to the agonist isoproterenol with an EC50 approximately 5-fold lower than that observed with the beta 1AR (0.53 +/- 0.14 versus 2.47 +/- 0.52 nM, p < 0.01). Deletion of the proline-rich sequence from the beta 1AR resulted in an improvement in isoproterenol-stimulated adenylyl cyclase to an EC50 value intermediate to that observed in the wild-type receptors (1.14 +/- 0.08 nM, p < 0.05 versus wild-type beta 1AR). In contrast, insertion of this sequence into the beta 2AR impaired its ability to mediate this process. Similar results were observed for receptor sequestration. Wild-type beta 1-and beta 2AR underwent maximal agonist-promoted sequestration of 25.9 +/- 4.0 and 60.0 +/- 3.3%, respectively. Deletion of the proline-rich region from the beta 1AR improved maximal sequestration to 43.0 +/- 2.7% (p < 0.01 versus wild-type beta 1AR), while insertion of the sequence into the beta 2AR impaired sequestration to 33.9 +/- 2.7% (p < 0.001 versus wild-type beta 2AR). We conclude that the distinct phenotypic patterns observed for these two agonist-promoted events in the beta 1AR and beta 2AR subtypes are partially due to the conformational effects of this proline-rich third intracellular loop sequence. Such regions, which are also found in some other G-protein-coupled receptors, may represent a general motif responsible for attenuating certain agonist-promoted receptor events.
β-肾上腺素能受体（βAR）亚型不仅在特征性配体亲和力方面存在差异，而且在介导激动剂促进的事件（如腺苷酸环化酶激活和受体隔离）的方式和程度上也有所不同。我们利用中国仓鼠成纤维细胞中的诱变和重组表达，来研究β1AR第三细胞内环中存在但β2AR中不存在的一段异常富含脯氨酸的24个氨基酸序列（PARPPSPSPSPVPAPAPPPGPPRP）对上述受体介导事件的影响。表达野生型β2AR的细胞对激动剂异丙肾上腺素刺激的腺苷酸环化酶反应，其半数有效浓度（EC50）比β1AR低约5倍（0.53±0.14对2.47±0.52 nM，p<0.01）。从β1AR中删除富含脯氨酸的序列，导致异丙肾上腺素刺激的腺苷酸环化酶活性提高，EC50值处于野生型受体观察值的中间水平（1.14±0.08 nM，与野生型β1AR相比p<0.05）。相反，将该序列插入β2AR会损害其介导此过程的能力。受体隔离也观察到类似结果。野生型β1AR和β2AR分别经历了最大激动剂促进的隔离，比例为25.
