Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:The role of amino acids in extracellular loops of the human P2Y1 receptor in surface expression and activation processes. 1032 57

Ligand recognition has been extensively explored in G protein-coupled A(1), A(2A), and A(2B) adenosine receptors but not in the A(3) receptor, which is cerebroprotective and cardioprotective. We mutated several residues of the human A(3) adenosine receptor within transmembrane domains 3 and 6 and the second extracellular loop, which have been predicted by previous molecular modeling to be involved in the ligand recognition, including His(95), Trp(243), Leu(244), Ser(247), Asn(250), and Lys(152). The N250A mutant receptor lost the ability to bind both radiolabeled agonist and antagonist. The H95A mutation significantly reduced affinity of both agonists and antagonists. In contrast, the K152A (EL2), W243A (6.48), and W243F (6.48) mutations did not significantly affect the agonist binding but decreased antagonist affinity by approximately 3-38-fold, suggesting that these residues were critical for the high affinity of A(3) adenosine receptor antagonists. Activation of phospholipase C by wild type (WT) and mutant receptors was measured. The A(3) agonist 2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine stimulated phosphoinositide turnover in the WT but failed to evoke a response in cells expressing W243A and W243F mutant receptors, in which agonist binding was less sensitive to guanosine 5'-gamma-thiotriphosphate than in WT. Thus, although not important for agonist binding, Trp(243) was critical for receptor activation. The results were interpreted using a rhodopsin-based model of ligand-A(3) receptor interactions.
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PMID:Identification by site-directed mutagenesis of residues involved in ligand recognition and activation of the human A3 adenosine receptor. 1189 Dec 21

P2Y receptors are G protein-coupled receptors stimulated by extracellular nucleotides. Both the P2Y(1) and the P2Y(6) receptors are preferentially activated by nucleoside 5'-diphosphates, but favor different base moieties. In the case of the P2Y(1) receptor the preferred base is adenine, while the P2Y(6) receptor is activated by uracil nucleotides. To identify potential amino acid domains that interact with the base moiety, we used a chimeric receptor approach, employing the human P2Y(1) receptor as core structure to investigate the role in receptor activation of extracellular loops (ELs) and transmembrane domains (TMs) of the rat P2Y(6) receptor. The chimeric receptors were expressed in COS-7 cells and measured for stimulation of phospholipase C (PLC) induced by the potent P2Y(1) receptor agonist 2-MeSADP or the potent P2Y(6) receptor agonist UDP. Replacement of the N-terminus or EL2 resulted in low ( approximately 50 microM) potency of the agonist 2-MeSADP, thus confirming the importance of EL2 in ligand recognition. Upon replacement of several regions, the potency of the P2Y(1) agonist 2-MeSADP was either 1-2 microM (N-terminus and EL1, or EL1 and EL3) or 72 microM (N-terminus and EL3). Concurrent replacement of three regions (N-terminus, EL1, and EL3) completely precluded activation by 2-MeSADP. Our study identified domains of the P2Y(6) receptor that contribute to receptor activation by UDP and hence seem to be involved in uracil recognition. Upon replacement with extracellular domains of the P2Y(6) receptor sequence we observed a trend toward gain of receptor-induced PLC activation by UDP, for example, in the chimera containing replacements of both the N-terminus and EL1. Exchange of three receptor domains led to a construct with an EC(50) value for UDP of 19 microM and a maximal inositol phosphate accumulation similar to the native P2Y(6) receptor. Within receptor constructs of combined domain exchanges the additional substitution of Tyr(110) by the corresponding Asn from the P2Y(6) receptor showed a significant increase for activation by UDP, but only when combined with the N-terminal domain and TM1. The residue Tyr(110) was identified to play an important role in the recognition of the nucleobase in the P2Y(1) and P2Y(6) receptors.
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PMID:Shift in purine/pyrimidine base recognition upon exchanging extracellular domains in P2Y 1/6 chimeric receptors. 1547 78