Effects of Deleting the Amino

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

GAN Xiao-Qing¹, WANG Ji-Yong¹, YANG
Qi-Heng¹, YU Qing-Ming², PEI Gang^2,3 and LI
Lin^1,3
( ¹Shanghai Institute of Biochemistry, the Chinese Academy of
Sciences, Shanghai 200031, China； ²Shanghai Institute of Cell Biology, the
Chinese Academy of Sciences, Shanghai 200031, China； ³Shanghai Research Center of Life
Sciences, Shanghai 200031, China )

Abstract    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 lacking 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, ³²⁹G-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.
Key Words    G protein-coupled receptor kinase
2(GRK-2)；
truncational mutation； amino-terminal domain； catalytic domain

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