Mutagenesis and peptide analysis of the DRY motif in the alpha2A adrenergic receptor: evidence for alternate mechanisms in G protein-coupled receptors.
In G protein-coupled receptors (GPCRs), a conserved aspartic acid in the DRY motif at the cytoplasmic end of helix 3 regulates the transition to the active state, while the adjacent arginine is crucial for G protein activation. To examine the functions of these two residues, we made D130I and R131Q mutations in the alpha2A adrenergic receptor (AR). We demonstrate that, unlike other GPCRs, the alpha2A AR is not constitutively activated by the D130I mutation, although the mutation increases agonist affinity. While the R131Q mutation severely disrupts function, it decreases rather than increasing agonist affinity as seen in other GPCRs. We then investigated the molecular effects of the same mutations in a peptide model and showed that Arg131 is not required for peptide-mediated G protein activation. These results indicate that the alpha2A AR does not follow the conventional GPCR mechanistic paradigm with respect to the function of the DRY motif.
Chung
Duane A
DA
Biophysics Research Division, The University of Michigan, Ann Arbor, MI 48109, USA.
Wade
Susan M
SM
Fowler
Carol B
CB
Woods
Danielle D
DD
Abada
Paolo B
PB
Mosberg
Henry I
HI
Neubig
Richard R
RR
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001667:Binding, Competitive; D000068438:Brimonidine Tartrate; D002384:Catalysis; D002460:Cell Line; D002942:Circular Dichroism; D000242:Cyclic AMP / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011810:Quinoxalines / Q000494:pharmacology; D011869:Radioligand Assay; D018341:Receptors, Adrenergic, alpha-2 / Q000737:chemistry* / Q000378:metabolism; D014162:Transfection
C547768:ADRA2A protein, human; D000316:Adrenergic alpha-Agonists; D010455:Peptides; D011810:Quinoxalines; D018341:Receptors, Adrenergic, alpha-2; D000068438:Brimonidine Tartrate; D001120:Arginine; D000242:Cyclic AMP; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins