Functional specificity of ADP-(P2Y12)-like receptors
Zusammenfassung der Projektergebnisse
Metabotropic pyrimidine- and purine-nucleotide receptors (P2Y receptors) belong to the superfamily of G protein-coupled receptors. Over the past decade, P2Y receptors have been cloned from a variety of tissues and species, and eight functional subtypes have been characterized. Most recently, members of the P2Y12-like receptor group, which includes the clopidogrel-sensitive ADP receptor, have been deorphanized. Receptors of the latter P2Y group are not only expressed in platelets but are also highly abundant in the central nervous system. The available functional data and their expression in neuronal and glial cells implicate their relevance in neurotransmission, microglial functions and reactive astrogliosis. The central goal of this project was to elucidate the function of P2Y12-like receptors at the molecular level (Part A, PI Schöneberg) and at the in vivo level (Part B, PI Schulz). The generation of a dynamic model, rather than a static snapshot of a crystal structure, describing the space for every amino acid within the receptor structure was one ambitious aim of the mutagenesis approach of part A. Using gene synthesis and regular mutagenesis techniques, more than 1,300 mutants were cloned and functionally characterized. Comparison of the in vitro results with an ortholog dataset of 77 sequences, representing evolutionary proofed in vivo function, revealed that the predictive value of ortholog sequences is very high (90%). This result clearly underlines the help of ortholog data to evaluate mutations found in patients. These data are much more relevant for new, less characterized proteins where functional data are momentarily not available. All experimental and ortholog data were implemented into a public P2Y12 3D-homology model showing the dynamic structure of this receptor. Additionally, taking advantage of constitutively active mutants, characterized in this project part, we screened a nucleotide library for agonists and inverse agonists of P2Y12. We could show that P2Y12 is not only an ADP, but also an ATP receptor. Modifications at the 3'-OH of the 2'-deoxyribose change the properties of ADP and ATP to inverse agonists, a clinically highly relevant feature. In part B of this project we addressed the functional relevance of the P2Y12-like receptor group in vivo by characterizing 4 knockout mouse models. For GPR82 deficiency, we found a metabolic phenotype. This receptor is involved in lipid metabolism and correlates with BMI in human populations. Its hypothalamic expression and function has to be studied in future. In depth-characterization of the P2Y14-deficient mouse model revealed that this receptor is a new modulator of proper insulin secretion. Transcriptome sequencing of wild-type and knockout pancreatic islets showed numerous genes differentially regulated in the knockout and known to play crucial roles in glucose homeostasis and insulin secretion. Another member of this subgroup of P2Y receptors, GPR34, plays a role in the immune system. Gene deficiency resulted in improper immune reaction. The high expression of this gene in microglia questioned its role in these cells. Here we could show that GPR34 deficiency results in morphological changes of microglia cells, detectable directly after birth. These microglia cells were not able to phagocyte endogenous material and pathogens as wildtype microglia. Transcriptome sequencing of microglia showed multiple pathways differentially regulated in gene-deficient microglia. Based on our results in part B we will further study the influence of P2Y12-like receptors in physiology and pathophysiology. Therefore, the results and open questions of the P2Y14 and GPR34 mouse models will help us to apply for DFG research grants.
Projektbezogene Publikationen (Auswahl)
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Altered immune response in mice deficient for the G protein-coupled receptor GPR34. J Biol Chem. 2010; 286: 2101–10
Liebscher I, Müller U, Teupser D, Engemaier E, Engel KM, Ritscher L, Thor D, Sangkuhl K, Ricken A, Wurm A, Piehler D, Schmutzler S, Fuhrmann H, Albert FW, Reichenbach A, Thiery J, Schöneberg T, Schulz A
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The ligand specificity of the G-protein-coupled receptor GPR34. Biochem J. 2010; 443: 841–50
Ritscher L, Engemaier E, Stäubert C, Liebscher I, Schmidt P, Hermsdorf T, Römpler H, Schulz A, Schöneberg T
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Reduced food intake and body weight in mice deficient for the G protein-coupled receptor GPR82. PLoS One. 2011; 6: e29400
Engel KM, Schröck K, Teupser D, Holdt LM, Tönjes A, Kern M, Dietrich K, Kovacs P, Krügel U, Scheidt HA, Schiller J, Huster D, Brockmann GA, Augustin M, Thiery J, Blüher M, Stumvoll M, Schöneberg T, Schulz A
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Using ortholog sequence data to predict the functional relevance of mutations in G-protein-coupled receptors. FASEB J. 2012; 26: 3273–81
Cöster M, Wittkopf D, Kreuchwig A, Kleinau G, Thor D, Krause G, Schöneberg T
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Identification of determinants required for agonistic and inverse agonistic ligand properties at the ADP receptor P2Y12. Mol Pharmacol. 2013; 83: 256–66
Schmidt P, Ritscher L, Dong EN, Hermsdorf T, Cöster M, Wittkopf D, Meiler J, Schöneberg T
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2014. The G protein-coupled receptor P2Y14 influences insulin release and smooth muscle function in mice. J Biol Chem. 2014; 289: 23353–66
Meister J, Le Duc D, Ricken A, Burkhardt R, Thiery J, Pfannkuche H, Polte T, Grosse J, Schöneberg T, Schulz A
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Altered microglial phagocytosis in GPR34-deficient mice. Glia. 2015; 63: 206–15
Preissler J, Grosche A, Lede V, Le Duc D, Krügel K, Matyash V, Szulzewsky F, Kallendrusch S, Immig K, Kettenmann H, Bechmann I, Schöneberg T, Schulz A
