To preserve homeostasis and proper function in every living organism, it is important for cells to communicate with each other and their environment. Cells are constantly processing a huge amount of extracellular stimuli through proteins called receptors. Receptors can transduce the signal from extracellular to intracellular compartments. G- protein coupled receptors are the biggest group, in which also belongs Cannabinoid receptor type 1 (CB1R). Endocannabinoid system regulates many biological processes such as learning, food intake, and movement. Obesity is a serious issue nowadays and in cases of claryfing its molecular-genetic basis, there was found Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1). SGIP1 has a role in the regulation of energetic balance and its overexpression is leading to a development of obesity. SGIP1 was detected as an interaction partner of CB1R and it had been found that it is involved in internalization via clathrin-mediated endocytosis (CME). Key proteins for initiation and early phase of CME are FCHO1/2, with which SGIP1 shares high sequential homology. However, effect of SGIP1 on internalization of activated CB1R is inhibitory unlike FCHO1/2,wheras detailed mechanism of its function remains unclear. The aim of this...

To preserve homeostasis and proper function in every living organism, it is important for cells to communicate with each other and their environment. Cells are constantly processing a huge amount of extracellular stimuli through proteins called receptors. Receptors can transduce the signal from extracellular to intracellular compartments. G-protein coupled receptors are the biggest group, in which also belongs Cannabinoid receptor type 1 (CB1R). Endocannabinoid system regulates many biological processes such as learning, food intake, and movement. Obesity is a serious issue nowadays and in cases of searching for candidate molecules, there was found Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1). SGIP1 has a role in the regulation of energetic balance and its overexpression is leading to a development of obesity. SGIP1 was detected as an interaction partner of CB1R and it had been found that it is involved in internalization via clathrin-mediated endocytosis. SGIP1 is very homological with FCHO1/2 - important proteins which participate on early stages of endocytosis. Mechanism of inhibitory effect of SGIP1 on internalization remains unclear. The aim of this study is to clarify the role of distinct domains of SGIP1 in context of endocytosis. Key...

The D1-class receptors (D1R, D5R) each possess distinct signaling characteristics; however, pharmacological selectivity between them remains elusive. The third intracellular loops (IL3) of D1R and D5R harbour divergent residues that may contribute to their individual signalling phenotypes. Here we probe the function of central region of IL3 of D1R and D5R using deletion mutagenesis. Radioligand binding and whole cell cAMP assays suggest that the N-terminal and C-terminal moieties of the central IL3 oppositely contribute to the constitutive and agonist-dependant activity of D1-Class receptors. Whereas the N-terminal deletions ablated constitutive activity and decreased DA-induced activation, C-terminal deletions induced robust increases. These data, interpreted in concert with structural predictions generated from homology modeling implicate the central IL3 as playing an important role in the activation and subtype-specific characteristics of the D1-class receptors. This study may serve as a basis for the development of novel drugs targeting the central IL3 region.

Acid-sensing ion channels (ASIC) are a class of ligand gated plasma membrane ion channels that are activated by low extracellular pH. During ischemia, ASIC1a are activated and contribute to the demise of neurons. Pharmacological block of ASIC1a provides neuroprotection at delayed time points. However, no endogenous receptors have been implicated in the modulation of ASIC1a activity. The hypothesis presented is that sigma receptor activation inhibits ASIC1a function and ASIC1a-induced [Ca²?]i elevations during acidosis and ischemia, which may be a mechanism by which sigma ligands provide neuroprotection following stroke. This hypothesis is based on the following observations: First, sigma receptors regulate multiple ion channels that become activated during ischemia. Second, ASIC1a remain functionally active hours beyond the ischemic insult and sigma receptors have been shown to be neuroprotective at delayed time points following stroke. Ratiometric Ca²+ fluorometry and whole-cell patch clamp experiments showed that sigma-1 receptor activation depresses elevations in [Ca²+]i and membrane currents mediated by ASIC1a channels in cortical neurons. Furthermore, most of the elevations in [Ca²+]i triggered by acidosis are the result of Ca²+ channels opening downstream of ASIC1a activation. Stimulation of sigma-1 receptors effectively suppressed these secondary Ca²+ fluxes both by inhibiting ASIC1a and the other channels directly. The signaling cascade linking sigma-1 receptors and ASIC1a was determined to involve a pertussis toxin-sensitive G protein and A-Kinase Anchoring Protein 150/calcineurin complex, which resulted in a decrease of acid-induced [Ca²+]i elevations and ASIC1a-mediated currents. Furthermore, immunohistochemical studies confirmed that sigma-1 receptors, ASIC1a and AKAP150 colocalize in the plasma membrane of cortical neuron cell bodies and in the dendritic processes of these cells. Additionally, concurrent exposure to acidosis and ischemia resulted in synergistic potentiation of [Ca²+]i dysregulation. Although ASIC1a activation does not induce long-lived priming of synaptic vesicles for release, channel activation does have a temporal effect on ischemia-mediated [Ca²+]i increases after ischemia onset. Moreover, presynaptic ASIC1a channels promote synaptic transmission during ischemia by overcoming block of neurotransmission and thus enhance postsynaptic [Ca²+]i elevations. Sigma-1 receptor activation decreased ischemia-mediated Ca²+ dysregulation at pH values of 7.4 - 6.0 and prevented the synergistic interaction between ischemia and acidosis.

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Kardiovaskuläre Ereignisse bilden die Haupttodesursache in den westlichen Ländern. Mit der Einführung von Clopidogrel, welches am ADP-Rezeptor P2Y12 wirkt, konnte die Mortalität und Morbidität von kardiovaskulären Ereignissen signifikant gesenkt werden. Der P2Y12 gehört als G-Protein-gekoppelter Rezeptor (GPCR) zur größten Gruppe membranständiger Rezeptoren, welche durch ihr ubiquitäres Vorkommen einen idealen Angriffspunkt in der Pharmakotherapie bilden. Zur intelligenten und gezielten Entwicklung von neuen Arzneimitteln bedarf es umfassender Kenntnisse der Struktur- und Wirkungsbeziehung von GPCR. Um den Modellrezeptor P2Y12 strukturell und funktionell zu charakterisieren, wurde eine sättigende Mutagenese in einem funktionell essentiellen Bereich des Rezeptors (Transmembranhelices 6 und 7 sowie 3. extrazellulärer Loop) durchgeführt. Hiermit sollten die Auswirkungen von Punktmutationen auf die Funktionsweise des Rezeptors untersucht werden. Hierfür wurden sättigende Mutantenbibliotheken für 66 Positionen erstellt, wobei jede Aminosäure (AS) durch jede nicht natürlicherweise im humanen P2Y12 vorkommende AS ersetzt wurde (1254 Mutanten). Diese wurden funktionell im Expressionssystem der Hefe Saccharomyces cerevisiae mit steigenden Agonistenkonzentrationen charakterisiert und anhand ihrer Funktionalität klassifiziert. Dabei wiesen 90,8 ± 1,9 % der Rezeptormutanten keine Wildtypeigenschaften auf. Die Auswertung von 77 Wirbeltierorthologen zeigte ebenso eine hohe Konservierung von 90,7 ± 1,5 % pro Position. Im direkten positionalen Vergleich zwischen evolutionären und in vitro Daten konnte eine Übereinstimmung der in vitro und in vivo Daten von 90,2 % gefunden werden. Die funktionellen Daten wurden in eine Online-Mutantendatenbank eingearbeitet und wurden in einem 3D-Rezeptor-Homologiemodell visualisiert. Damit ist der Beweis geführt worden, dass es mit guter Vorhersagewahrscheinlichkeit möglich ist, von evolutionären Daten Rückschlüsse auf die Relevanz von Mutationen zu ziehen.

Protein-protein interaction dominates all major biology processes in living cells. Recent studies suggestthat the surface expression and activity of G protein-coupled receptors (GPCRs), which are the largestfamily of receptors in human cells, can be modulated by receptor activity–modifying proteins (RAMPs). Computational tools are essential to complement experimental approaches for the understanding ofmolecular activity of living cells and molecular dynamics simulations are well suited to providemolecular details of proteins function and structure. The classical atom-level molecular modeling ofbiological systems is limited to small systems and short time scales. Therefore, its application iscomplicated for systems such as protein-protein interaction in cell-surface membrane. For this reason, coarse-grained (CG) models have become widely used and they represent an importantstep in the study of large biomolecular systems. CG models are computationally more effective becausethey simplify the complexity of the protein structure allowing simulations to have longer timescales. The aim of this degree project was to determine if the applications of coarse-grained molecularsimulations were suitable for the understanding of the dynamics and structural basis of the GPCRRAMP interactions in a membrane environment. Results indicate that the study of protein-proteininteractions using CG needs further improvement with a more accurate parameterization that will allowthe study of complex systems.

Flies are a widely distributed pest insect that poses a significant threat to food security. Flight is essential for the dispersal of the adult flies to find new food sources and ideal breeding spots. The supply of metabolic fuel to power the flight muscles of insects is regulated by adipokinetic hormones (AKHs). The fruit fly, Drosophila melanogaster, the flesh fly, Sarcophaga crassipalpis, and the oriental fruit fly, Bactrocera dorsalis all have the same AKH that is present in the blowfly, Phormia terraenovae; this AKH has the code-name Phote-HrTH. Binding of the AKH to the extracellular binding site of a G protein-coupled receptor causes its activation. In this thesis, the structure of Phote-HrTH in SDS micelle solution was determined using NMR restrained molecular dynamics. The peptide was found to bind to the micelle and be reasonably rigid, with an S 2 order parameter of 0.96. The translated protein sequence of the AKH receptor from the fruit fly, Drosophila melanogaster, the flesh fly, Sarcophaga crassipalpis, and the oriental fruit fly, Bactrocera dorsalis were used to construct two models for each receptor: Drome-AKHR, Sarcr-AKHR, and Bacdo-AKHR. It is proposed that these two models represent the active and inactive state of the receptor. The models based on the crystal structure of the β-2 adrenergic receptor were found to bind Phote-HrTH with a predicted binding free energy of –107 kJ mol–1 for Drome-AKHR, –102 kJ mol–1 for Sarcr-AKHR and –102 kJ mol–1 for Bacdo-AKHR. Under molecular dynamics simulation, in a POPC membrane, the β-2AR receptor-like complexes transformed to rhodopsin-like. The identification and characterisation of the ligand-binding site of each receptor provide novel information on ligand-receptor interactions, which could lead to the development of species-specific control substances to use discriminately against these pest flies.

Heterotrimeric Gi proteins play a role in signalling activated by lipopolysaccharide (LPS), Staphylococcus aureus (SA) and group B streptococci (GBS), leading to production of inflammatory mediators. We hypothesized that genetic deletion of Gi proteins would alter cytokine and chemokine production induced by LPS, SA and GBS stimulation. LPS-induced, heat-killed SA-induced and heat-killed GBS-induced cytokine and chemokine production in peritoneal macrophages from wild-type (WT), Gαi2-/- or Gαi1/3-/- mice were investigated. LPS induced production of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-10 and interferon-γ-inducible protein-10 (IP-10); SA induced TNF-α, and IL-1β production; and GBS induced TNF-α, IL-6, IL-1β, macrophage inflammatory protein-1α (MIP-1α) and keratinocyte chemoattract (KC) production were all decreased (P < 0.05) in Gαi2-/- or Gαi1/3-/- mice compared with WT mice. In contrast to the role of Gi proteins as a positive regulator of mediators, LPS-induced production of MIP-1α and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in macrophages from Gαi1/3-/- mice, and SA-induced MIP-1α production was increased in both groups of Gαi protein-depleted mice. LPS-induced production of KC and IL-1β, SA-induced production of GM-CSF, KC and IP-10, and GBS-induced production of IL-10, GM-CSF and IP-10 were unchanged in macrophages from Gαi2-/- or Gαi1/3-/- mice compared with WT mice. These data suggest that Gi2 and Gi1/3 proteins are both involved and differentially regulate murine inflammatory cytokine and chemokine production in response to both LPS and Gram-positive microbial stimuli.

We have shown previously [Hosohata, K., Logan, J.K., Varga, E., Burkey, T.H., Vanderah, T.W., Porreca, F., Hruby, V.J., Roeske, W.R., Yamamura, H.I., 2000. The role of the G protein γ2 subunit in opioid antinociception in mice. Eur. J. Pharmacol. 392, R9-R11] that intracerebroventricular (i.c.v.) treatment of mice with a phosphorothioate oligodeoxynucleotide antisense to the γ2 subunit (Gγ2) of the heterotrimeric G proteins (antisense ODN) significantly attenuates antinociception by a δ-opioid receptor agonist. In the present study, we examined the involvement of Gγ2 in antinociception mediated by other (μ- or κ-opioid, cannabinoid, α2-adrenoreceptor) analgesic agents in a warm (55°C) water tail-flick test in mice. Interestingly, i.c.v. treatment with the antisense ODN attenuated antinociception by each analgesic agent. Missense phosphorothioate oligodeoxynucleotide treatment, on the other hand, had no effect on antinociception mediated by these agonists. The antinociceptive response recovered in 6 days after the last antisense ODN injection, indicating a lack of nonspecific tissue damage in the animals. These results suggest a pervasive role for the G protein γ2 subunits in supraspinal antinociception.