Liang W, Bidwell C A, Collodi P R, Mills S E
Department of Animal Sciences, Purdue University, West Lafayette, IN 47907, USA.
J Anim Sci. 2000 Sep;78(9):2329-35. doi: 10.2527/2000.7892329x.
The gene for the porcine beta2-adrenergic receptor (pbeta2AR) was transfected into Chinese hamster ovary (CHO) cells for expression. Fourteen stable cell lines were obtained and exhibited receptor densities ranging from 12 to 2,371 fmol/mg membrane protein. The receptor density was not correlated with estimates of gene copy number obtained by Southern hybridization. The pbeta2AR in CHO cells exhibited saturable binding of [125I]CYP (Kd = 14.5 pM) and stereospecificity for (-)- and (+)-isoproterenol. The relative affinities for (-)-isoproterenol (ISO), (-)-epinephrine (EPI), and (-)-norepinephrine (NEPI) were ISO > EPI > NEPI, which are characteristic of beta2AR. The affinity values for these ligands were similar to those in other species. Binding of ISO, EPI, and NE revealed two affinity states of the betaAR; the high-affinity state was eliminated by adding Gpp(NH)p, a nonhydrolyzable GTP analogue. Binding of the antagonist propranolol modeled to only one affinity state, and Gpp(NH)p did not affect binding. Multiple affinity states are characteristic of agonist-induced coupling of betaAR with G-proteins, and the data suggest that the cloned pbetaAR is functionally competent. Data confirm that the pbeta2AR is the pig version of beta2AR. Stable CHO cell lines will be useful for characterization of pbeta2AR and screening and designing potential drugs that may be used to enhance pig production.
将猪β2 - 肾上腺素能受体(pβ2AR)基因转染至中国仓鼠卵巢(CHO)细胞中进行表达。获得了14个稳定细胞系,其受体密度范围为12至2371 fmol/mg膜蛋白。受体密度与通过Southern杂交获得的基因拷贝数估计值不相关。CHO细胞中的pβ2AR表现出对[125I]CYP的饱和结合(Kd = 14.5 pM)以及对( - ) - 和( + ) - 异丙肾上腺素的立体特异性。对( - ) - 异丙肾上腺素(ISO)、( - ) - 肾上腺素(EPI)和( - ) - 去甲肾上腺素(NEPI)的相对亲和力为ISO>EPI>NEPI,这是β2AR的特征。这些配体的亲和力值与其他物种中的相似。ISO、EPI和NE的结合揭示了βAR的两种亲和力状态;通过添加不可水解的GTP类似物Gpp(NH)p消除了高亲和力状态。拮抗剂普萘洛尔的结合仅模拟为一种亲和力状态,并且Gpp(NH)p不影响结合。多种亲和力状态是激动剂诱导的βAR与G蛋白偶联的特征,数据表明克隆的pβAR在功能上是有活性的。数据证实pβ2AR是猪版的β2AR。稳定的CHO细胞系将有助于表征pβ2AR以及筛选和设计可能用于提高猪生产性能的潜在药物。