Key determinants of nucleotide-activated G protein-coupled P2Y(2) receptor function revealed by chemical and pharmacological experiments, mutagenesis and homology modeling.
The P2Y(2) receptor, which is activated by UTP, ATP, and dinucleotides, was studied as a prototypical nucleotide-activated GPCR. A combination of receptor mutagenesis, determination of its effects on potency and efficacy of agonists and antagonists, homology modeling, and chemical experiments was applied. R272 (extracellular loop EL3) was found to play a gatekeeper role, presumably responsible for recognition and orientation of the nucleotides. R272 is also directly involved in binding of dinucleotides, which behaved as partial agonists. Y118A (3.37) mutation led to dramatically reduced efficacy of agonists; it is part of the entry channel as well as the triphosphate binding site. While the Y114A (3.33) mutation did not have any effect on agonist activities, the antagonist Reactive Blue 2 (6) was completely inactive at that mutant. The disulfide bridge Cys25-Cys278 was found to be important for agonist potency but neither for agonist efficacy nor for antagonist potency.
PharmaCenter Bonn, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany.
Petra
P
Hillmann
Geun-Yung
GY
Ko
Andreas
A
Spinrath
Alexandra
A
Raulf
Ivar
I
von Kügelgen
Samuel C
SC
Wolff
Robert A
RA
Nicholas
Evi
E
Kostenis
Hans-Dieter
HD
Höltje
Christa E
CE
Müller
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D004220:Disulfides / Q000737:chemistry; D015195:Drug Design; D004797:Enzyme-Linked Immunosorbent Assay; D005110:Extracellular Space / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000494:pharmacology*; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D018048:Receptors, Purinergic P2 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D004220:Disulfides; D008024:Ligands; D009711:Nucleotides; C546385:P2RY2 protein, human; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2