Establishment of Functional Analysis by Expressing Antibody Molecule in the Cells

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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.