Abstract
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 </=100 microM 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.
Highlights
P2 receptors have been divided into two structurally distinct families as follows: the P2X receptor class of ligand-gated ion channels, which are primarily activated by ATP, and the P2Y receptor class of G protein-coupled receptors (GPCRs),1 which are activated by both extracellular adenine and uridine nucleotides [1,2,3]
We have investigated the determinants of ligand recognition in P2Y1 receptors using site-directed mutagenesis of specific amino acid residues in the transmembrane helical domains (TMs) [5], molecular modeling based on similarity to rhodopsin in sequence and overall geometry [6], and the chemical synthesis of novel agonists [7] and antagonists [8]
In the present study we have identified both charged residues and Cys residues within the extracellular loops (ELs) of the human P2Y1 receptor that are critical for the activation of the receptor
Summary
P2 receptors have been divided into two structurally distinct families as follows: the P2X receptor class of ligand-gated ion channels, which are primarily activated by ATP, and the P2Y receptor class of G protein-coupled receptors (GPCRs), which are activated by both extracellular adenine and uridine nucleotides [1,2,3]. Most of the interest in GPCRs to date has focused on the TM domains for locating amino acids that are involved in recognition of small, non-peptide ligands, including nucleotides [5, 9, 10]. A conserved disulfide bridge between Cys residues in the second extracellular loop (EL2) and the exofacial end of TM3 has been shown to be essential for various biogenic amine and other GPCRs. It is generally thought that this disulfide bond is required to maintain overall receptor geometry, its role in transport to the cell surface is contradictory [18, 19]. P2Y receptors might form two disulfide bridges within the extracellular receptor portions
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
