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-coupled receptor kinases (GRKs), two amino terminus-truncated mutants (delta30 or delta15) and two single-amino-acid mutants of conserved acidic residues (D2A or E7A) of human GRK1 were constructed and expressed in human embryonic kidney 293 cells. It was shown that truncated mutations and one single-point mutation (E7A) greatly decreased GRK1's activity to phosphorylate photoactivated rhodopsin (Rho*), whereas the abilities of these mutants to phosphorylate 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 (delta30 or delta15) of GRK2, representative of another GRK subfamily, also abolished the kinase's activity toward Rho*. The similar single-point mutation (E5A) of GRK2 heavily impaired its phosphorylation of Rho* but did not alter its ability to phosphorylate the peptide, and the G329-rhodopsin-augmented peptide phosphorylation 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.
Yu
Q M
QM
Shanghai Institute of Cell Biology, Chinese Academy of Sciences, China.
Cheng
Z J
ZJ
Gan
X Q
XQ
Bao
G B
GB
Li
L
L
Pei
G
G
D000217:Acyltransferases / Q000378:metabolism*; D000595:Amino Acid Sequence; D015153:Blotting, Western; D017124:Conserved Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D019204:GTP-Binding Proteins / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D012243:Rhodopsin / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D014162:Transfection; D014466:Ultraviolet Rays; D051552:beta-Adrenergic Receptor Kinases
D005136:Eye Proteins; D018698:Glutamic Acid; D012243:Rhodopsin; D000217:Acyltransferases; C018764:2-acylglycerophosphate acyltransferase; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; C496764:GRK1 protein, human; D051552:beta-Adrenergic Receptor Kinases; D019204:GTP-Binding Proteins