Falck-Pedersen E, Heinflink M, Alvira M, Nussenzveig D R, Gershengorn M C
Department of Microbiology, Cornell University Medical College, New York, New York 10021.
Mol Pharmacol. 1994 Apr;45(4):684-9.
Biological studies of seven-transmembrane region G protein-coupled receptors have been restricted by available techniques for gene transfer into mammalian cells. We have created a highly efficient adenovirus-based expression vector for the thyrotropin-releasing hormone (TRH) receptor (TRH-R), AdCMVmTRHR, to circumvent difficulties encountered when transient or stable plasmid expression systems are used. We show that infection with AdCMVmTRHR results in fully functional TRH-Rs, which can be expressed in a broad range of mammalian cell types, including those resistant to conventional transient transfection. TRH-Rs can be expressed at high levels, up to 2 x 10(6) receptors/cell. Expression in several cell lines in culture reveals that rapid TRH-R desensitization by TRH and phorbol 12-myristate 13-acetate is cell type specific. The versatility of adenovirus-mediated gene transfer and expression of TRH-Rs not only facilitates in vitro studies of TRH-R biology but also provides a valuable in vivo expression vector capable of extending TRH-R studies to animal model systems.
七跨膜区G蛋白偶联受体的生物学研究一直受到现有基因导入哺乳动物细胞技术的限制。我们构建了一种高效的基于腺病毒的促甲状腺激素释放激素(TRH)受体(TRH-R)表达载体AdCMVmTRHR,以克服使用瞬时或稳定质粒表达系统时遇到的困难。我们发现,用AdCMVmTRHR感染可产生功能完全正常的TRH-R,其可在多种哺乳动物细胞类型中表达,包括那些对传统瞬时转染有抗性的细胞。TRH-R可高水平表达,最高可达2×10⁶受体/细胞。在多种培养细胞系中的表达显示,TRH和佛波醇12-肉豆蔻酸酯13-乙酸酯对TRH-R的快速脱敏具有细胞类型特异性。腺病毒介导的TRH-R基因转移和表达的多功能性不仅有助于TRH-R生物学的体外研究,还提供了一种有价值的体内表达载体,能够将TRH-R研究扩展到动物模型系统。