Effects of Deleting the Amino-terminal Domain of GRK-2 on Its Function.

To reveal the possible role of the amino terminal domain of G protein coupled receptor kinases(GRKs)in receptor phosphorylation and/or modulation of its kinase activity, a truncated mutant of GRK 2 lac king the amino terminal domain(ΔN GRK2)was made. ΔN GRK2 was expressed effectively in E.coli as a GST fusion protein and was purified by affinity chromatography on a GSH Sepharose column. ΔN GRK2 was then separated from GST tag by thrombin cleavage and recovered. Although ΔN GRK2 had nearly identical activity with wild type GRK 2 in phosphorylation of peptide substrate, it completely lost the ability to phosphorylate the light activated receptor rhodopsin. Furthermore, deletion of the amino terminal domain rendered GRK 2 unresponsive to the regulation of kinase activity by a truncated form of rhodopsin, 329 G Rho * and βγ subunits of G protein. These results demonstrated that the amino terminal domain was necessary to GRK2 for both the phosphorylation of receptor and the regulation of its kinase activity by the receptor. It was reasonable to postulate that this domain has little, if any effect on the catalytic domain of natural form of GRK2.