The amino terminus with a conserved glutamic acid of G protein-coupled receptor kinases is indispensable for their ability to phosphorylate photoactivated rhodopsin

To investigate functions of the consensus amino terminus of G protein-coupl ed receptor kinases (GRKs), two amino terminus-truncated mutants (Delta 30 or Delta 15) and two single-amino-acid mutants of conserved acidic residues (D2A or E7A) of human GRK1 were constructed and expressed in human embryon ic kidney 293 cells. It was shown that truncated mutations and one single-p oint mutation (E7A) greatly decreased GRK1's activity to phosphorylate phot oactivated rhodopsin (Rho*), whereas the abilities of these mutants to phos phorylate a synthetic peptide substrate and to translocate from cytosol to rod outer segments on light activation were unaffected. Further experiments demonstrated that the same truncated mutations (Delta 30 or Delta 15) of G RK2, representative of another GRK subfamily, also abolished the kinase's a ctivity toward Rho*. The similar single-point mutation (E5A) of GRK2 heavil y impaired its phosphorylation of Rho* but did not alter its ability to pho sphorylate the peptide, and the G(329)-rhodopsin-augmented peptide phosphor ylation by GRK2 (E5A) remained unchanged. Our data, taken together, suggest that the amino terminus as well as a conserved glutamic acid in the region of GRKs appears essential for their ability to functionally interact with G protein-coupled receptors.