Farrar S J, Whiting P J, Bonnert T P, McKernan R M
Department of Biochemistry and Molecular Biology, Merck Sharp and Dohme Research Laboratories, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, United Kingdom.
J Biol Chem. 1999 Apr 9;274(15):10100-4. doi: 10.1074/jbc.274.15.10100.
We have developed a method to determine the stoichiometry of subunits within an oligomeric cell surface receptor using fluorescently tagged antibodies to the individual subunits and measuring energy transfer between them. Anti-c-Myc monoclonal antibody (mAb 9-E10) derivatized with a fluorophore (europium cryptate, EuK) was used to individually label c-Myc-tagged alpha1-, beta2-, or gamma2-subunits of the hetero-oligomeric gamma-aminobutyric acid (GABAA) receptor in intact cells. The maximal fluorescent signal derived from the alpha1(c-Myc)beta2gamma2 and the alpha1beta2(c-Myc)gamma2 receptors was twice that obtained with alpha1beta2gamma2(c-Myc), suggesting that there are 2x alpha-, 2x beta-, and 1x gamma-subunits in a receptor monomer. This observation was extended using fluorescence energy transfer. Receptors were half-maximally saturated with EuK-anti-c-Myc mAb, and the remaining alpha1(c-Myc) subunits were labeled with excess anti-c-Myc mAb derivatized with the fluorescence energy acceptor, XL665. On exposure to laser light, energy transfer from EuK to XL665 occurred with alpha1(c-Myc)beta2gamma2 and alpha1beta2(c-Myc)gamma2, but no significant energy transfer was observed with alpha1beta2gamma2(c-Myc) receptors, indicating the absence of a second gamma-subunit in a receptor monomer. We confirm that the GABAA receptor subtype, alpha1beta2gamma2, is composed of two copies each of the alpha- and beta-subunits and one copy of the gamma-subunit (i.e. (alpha1)2(beta2)2(gamma2)1) and conclude that this method would have general applicability to other multisubunit cell surface proteins.
我们开发了一种方法，通过使用针对各个亚基的荧光标记抗体并测量它们之间的能量转移，来确定寡聚细胞表面受体内亚基的化学计量。用荧光团（铕穴状化合物，EuK）衍生化的抗c-Myc单克隆抗体（mAb 9-E10）用于在完整细胞中单独标记异源寡聚γ-氨基丁酸（GABAA）受体的c-Myc标记的α1-、β2-或γ2-亚基。源自α1(c-Myc)β2γ2和α1β2(c-Myc)γ2受体的最大荧光信号是α1β2γ2(c-Myc)的两倍，这表明受体单体中有2个α-亚基、2个β-亚基和1个γ-亚基。利用荧光能量转移扩展了这一观察结果。用EuK-抗c-Myc mAb使受体达到半数最大饱和，其余的α1(c-Myc)亚基用过量的用荧光能量受体XL665衍生化的抗c-Myc mAb进行标记。在激光照射下，α1(c-Myc)β2γ2和α1β2(c-Myc)γ2发生了从EuK到XL665的能量转移，但α1β2γ2(c-Myc)受体未观察到明显的能量转移，这表明受体单体中不存在第二个γ-亚基。我们证实GABAA受体亚型α1β2γ2由α-和β-亚基各两个拷贝以及γ-亚基一个拷贝组成（即(α1)2(β2)2(γ2)1），并得出结论，该方法对其他多亚基细胞表面蛋白具有普遍适用性。
