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J Biol Chem, Vol. 274, Issue 21, 14639-14647, May 21, 1999
From the The P2Y1 receptor is a
membrane-bound G protein-coupled receptor stimulated by adenine
nucleotides. Using alanine scanning mutagenesis, the role in receptor
activation of charged amino acids (Asp, Glu, Lys, and Arg) and
cysteines in the extracellular loops (EL) of the human P2Y1
receptor has been investigated. The mutant receptors were expressed in
COS-7 cells and measured for stimulation of phospholipase C induced by
the potent agonist 2-methylthioadenosine-5'-diphosphate (2-MeSADP). In
addition to single point mutations, all receptors carried the
hemagglutinin epitope at the N- terminus for detection of cell-surface
expression. The C124A and C202A mutations, located near the exofacial
end of transmembrane helix 3 and in EL2, respectively, ablated
phospholipase C stimulation by
The Role of Amino Acids in Extracellular Loops of the Human
P2Y1 Receptor in Surface Expression and Activation
Processes
,,
Molecular Recognition Section, Laboratory of
Bioorganic Chemistry, NIDDK, National Institutes of Health,
Bethesda, Maryland 20892 and the § Department of
Pharmacology, University of North Carolina School of Medicine,
Chapel Hill, North Carolina 27599
100 µM 2-MeSADP. Surface enzyme-linked immunosorbent assay detection of both mutant receptors showed <10% expression, suggesting that a critical disulfide bridge between EL2 and the upper part of transmembrane 3, as found in many
other G protein-coupled receptors, is required for proper trafficking
of the P2Y1 receptor to the cell surface. In contrast, the
C42A and C296A mutant receptors (located in the N-terminal domain and
EL3) were activated by 2-MeSADP, but the EC50 values were
>1000-fold greater than for the wild-type receptor. The double mutant
receptor C42A/C296A exhibited no additive shift in the concentration-response curve for 2-MeSADP. These data suggest that
Cys42 and Cys296 form another disulfide bridge
in the extracellular region, which is critical for activation.
Replacement of charged amino acids produced only minor changes in
receptor activation, with two remarkable exceptions. The E209A mutant
receptor (EL2) exhibited a >1000-fold shift in EC50.
However, if Glu209 were substituted with amino acids
capable of hydrogen bonding (Asp, Gln, or Arg), the mutant receptors
responded like the wild-type receptor. Arg287 in EL3 was
impaired similarly to Glu209 when substituted by alanine.
Substitution of Arg287 by lysine, another positively
charged residue, failed to fully restore wild-type activity.
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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