Establishment of Functional Analysis by Expressing Antibody Molecule in the Cells

为了分析6种G蛋白偶联受体激酶在细胞内的功能差异，我们试图建立一种新的方法，即细胞内免疫。细胞内免疫是一种表达单抗的功能性灭活方法。首先，将谷胱甘肽-S-转移酶融合蛋白与β-ARK1的羧基末端免疫，制备了与GRK2(β-ARK1)反应的单抗。Western印迹结果表明，该单抗能特异性识别βARK1，但不能识别GRK3、5和6。用硫酸铵沉淀法和Protein G柱层析法从杂交瘤细胞培养上清液中纯化单抗。我们发现，纯化的mAb抑制了基础的和激动剂刺激的βARK1的磷酸化活性。该单抗还能抑制异三聚体G蛋白与β-ARK1羧基末端的β-氨基结合。由于GST-羧基末端融合蛋白能够刺激BetaARK1的磷酸化活性，我们认为抗体结合在该部分是活性所必需的，并抑制了BetaARK1的激活。我们还将单抗注射到心肌细胞中，发现β_1-肾上腺素能受体参与了β_1肾上腺素能受体介导的L钙通道的调节过程。为构建单链抗体分子(ScFv)在细胞中表达mAb基因，从杂交瘤组织中扩增出mAb重链和轻链可变区，并在大肠杆菌中表达。表达的单链抗体能识别βARK1。我们还将scFv连接到粘粒载体上，制成重组腺病毒。我们认为，腺病毒介导的ScFv的表达有助于阐明BetaARK1在各种G蛋白偶联受体脱敏中的作用。

To analyze the functional differences among six G protein-coupled receptor kinases (GRKs) in the cells, we tried to establish a novel method, i.e.intracellular immunization. The intracellular immunization is a functional inactivation method by expressing monoclonal antibody (mAb). At first, mAb that reacts with GRK2 (betaARK1) was made by immunizing gluthatione-S-transferase (GST) fusion protein with carboxyl terminus of betaARK1. The resulting mAb specifically recognized betaARKl but not GRK3,5 and 6 with Western blot. The mAb was purified from culture supenatant of hybridoma by ammonium sulfate precipitation and Protein G column. We found that the purified mAb inhibited the basal and agonist-stimulated phosphorylating activities of betaARK1. The mAb also inhibited thc betagammabinding of heterotrimeric G protein to carboxyl terminus of betaARK1. As the GST-carboxyl terminus fusion protein could stimulate the phosphorylating activity of betaARKl, we concluded that antibody bound to the part to be essential for the activity and inhibited the activation of betaARKl. We have also injected mAb into myocytes and found the involvement of betaARKl in the process of beta _1 -adrenergic receptor-mediated modulation of L-type Ca ^<2+> channel. To construct single chain Fv molecule (scFv) to express mAb gene in the cell, the variable regions of heavy and light chains of mAb was amplified from mRNA of hybridoma by PCR.The scFv was expressed in E.coli. and the expressed scFv could recognize the betaARKl. We also ligated scFv into a cosmid vector to make a recombinant adenovirus. We believe that adenovirus-mediated expression of scFv can help to elucidate the roles of betaARKl in desensitization of various G protein-coupled receptors.