Biomedical subjects

Mark H Pausch

Publications and source records attributed to Mark H Pausch.

5 recordsLinked to original sources

PubMed2006

Aplindore (DAB-452), a high affinity selective dopamine D2 receptor partial agonist.

The pharmacology of aplindore (DAB-452) was characterized in CHO-K1 cells stably transfected with the human dopamine D(2) receptor short isoform (CHO-D(2s)) and in a behavioral model for post-synaptic agonism in rats. In [(3)H]-spiperone competition binding studies, aplindore showed high affinity for dopamine D(2) and D(3) receptors and low affinity for the dopamine D(4), serotonin (5-HT)(1A), 5-HT(2) receptors and the alpha1-adrenoceptor. The high potency partial agonist activity of aplindore was demonstrated in [(35)S]guanosine 5'-O-(3-thiotriphosphate) ([(35)S]GTPgammaS) binding, extracellular signal-regulated kinase (ERK)-phosphorylation and intracellular calcium flux assay using fluorometric plate reader ([Ca(2+)](i)-FLIPR) format. The [Ca(2+)](i)-FLIPR assay was conducted with CHO-D(2S) receptor cells also stably expressing chimeric G(alphaq/o)-proteins. In all assay modalities, the potencies and intrinsic activities of aplindore were lower than dopamine and higher than aripiprazole. In contrast to the [(35)S]GTPgammaS binding and ERK-phosphorylation assays, the [Ca(2+)](i)-FLIPR assay was able to detect the low partial agonist activity of SDZ 208-912. In unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats, aplindore induced contralateral turning, which was blocked by the dopamine D(2) receptor antagonist raclopride. The dopamine D(2) receptor selective partial agonist profile of aplindore suggests that it should be effective for the treatment of dopaminergic-based disorders, such as schizophrenia and Parkinson's disease.

Animals↗

PubMed2006

Characterization of Gpr101 expression and G-protein coupling selectivity.

This report describes the identification and characterization of the murine orphan GPCR, Gpr101. Both human and murine genes were localized to chromosome X. Similar to its human ortholog, murine Gpr101 mRNA was detected predominantly in the brain within discrete nuclei. A knowledge-restricted hidden Markov model-based algorithm, capable of accurately predicting G-protein coupling selectivity, indicated that both human and murine GPR101 were likely coupled to Gs. This prediction was supported by the elevation of cyclic AMP levels and the activation of a cyclic AMP response element-luciferase reporter gene in HEK293 cells over-expressing human GPR101. Consistent with this, over-expression of human GPR101 in a yeast-based system yielded an elevated, agonist-independent reporter gene response in the presence of a yeast chimeric Galphas protein. These results indicate that GPR101 participates in a potentially wide range of activities in the CNS via modulation of cAMP levels.

Animals↗

PubMed2004

Functional expression of human and mouse P2Y12 receptors in Saccharomyces cerevisiae.

DNA sequences encoding the murine ortholog of the human P2Y12 receptor were cloned. The human and mouse P2Y12 receptors were expressed in a yeast cell-based GPCR expression technology containing chimeric yeast Galpha protein (Gpa1) constructs in which the 5 C-terminal amino acids were identical to corresponding sequences from mammalian Galphai/o proteins. LacZ reporter gene assays of agonist-induced activation of the G protein-coupled mating signal transduction pathway revealed murine P2Y12 functional pharmacological properties that closely resembled those exhibited by the human P2Y12 receptor. In NIH3T3 cells, the mouse P2Y12 stimulated calcium uptake monitored in FLIPR via coupling to a Galphaq/i3 chimeric protein. Murine P2Y12 mRNA was expressed at high levels in the brain and at lower levels in a variety of peripheral tissues. In situ hybridization analysis indicated glia-specific expression within the brain.

Amino Acid Sequence↗

PubMed2004

Predicting GPCR-G-protein coupling using hidden Markov models.

MOTIVATION: Determining the coupling specificity of G-protein coupled receptors (GPCRs) is important for understanding the biology of this class of pharmacologically important proteins. Currently available in silico methods for predicting GPCR-G-protein coupling specificity have high error rate. METHOD: We introduce a new approach for creating hidden Markov models (HMMs) based on a first guess about the importance of various residues. We call these knowledge restricted HMMs to emphasize the fact that the state space of the HMM is restricted by the application of a priori knowledge. Specifically, we use only those amino acid residues of GPCRs which are likely to interact with G-proteins, namely those that are predicted to be in the intra-cellular loops. Furthermore, we concatenate these predicted loops into one sequence rather than considering them as four disparate units. This reduces the HMM state space by drastically decreasing the sequence length. RESULTS: Our knowledge restricted HMM based method to predict GPCR-G-protein coupling specificity has an error rate of <1%, when applied to a test set of GPCRs with known G-protein coupling specificity. AVAILABILITY: Academic users can get the data set mentioned herein and HMMs from the authors.

Binding Sites↗

PubMed2003

Development of a yeast bioassay to characterize G protein-coupled receptor kinases. Identification of an NH2-terminal region essential for receptor phosphorylation.

G protein-coupled receptor kinases (GRKs) specifically bind and phosphorylate the agonist-occupied form of G protein-coupled receptors. To further characterize the mechanism of GRK/receptor interaction, we developed a yeast-based bioassay using strains engineered to functionally express the somatostatin receptor subtype 2 and exhibit agonist-dependent growth. Here, we demonstrate that agonist-promoted growth was effectively inhibited by co-expression with either wild type GRK2 or GRK5, whereas catalytically inactive forms of these kinases were without effect. In an effort to identify residues involved in receptor interaction, we generated a pool of GRK5 mutants and then utilized the bioassay to identify mutants selectively deficient in inhibiting agonist-promoted growth. This resulted in the identification of a large number of mutants, several of which were expressed, purified, and characterized in more detail. Two of the mutants, GRK5-L3Q/K113R and GRK5-T10P, were defective in receptor phosphorylation and also exhibited a partial defect in phospholipid binding and phospholipid-stimulated autophosphorylation of the kinase. In contrast, these mutants had wild type activity in phosphorylating the non-receptor substrate tubulin. To further characterize the function of the NH2-terminal region of GRK5, we generated a deletion mutant lacking residues 2-14 and found that this mutant was also severely impaired in receptor phosphorylation and phospholipid-promoted autophosphorylation. In addition, an NH2-terminal 14-amino acid peptide from GRK5 selectively inhibited receptor phosphorylation by GRK5 but had minimal effect on GRK2 activity. Based on these findings, we propose a model whereby the extreme NH2 terminus of GRK5 mediates phospholipid binding and is required for optimal receptor phosphorylation.

Animals↗