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A study of molecular interactions of the μ-opioid receptor: the effect of biased ligands
Marková, Vendula ; Novotný, Jiří (advisor) ; Rudajev, Vladimír (referee)
G protein-coupled receptors (GPCRs) are the largest group of membrane-bound receptors. Transmission of signals into the cell interior is mediated through the interactions of these receptors with other signaling molecules. Nowadays, a great attention is devoted to biased ligands which are able to alter the conformation of the receptor in a specific way and thus distinctly affect its function. This diploma thesis was focused on a study of µ-opioid receptor (MOR), which is important in nociception. The aim of this study was to find out, how the activation of MOR by specific biased ligands (morphine, endomorphin-2 and DAMGO) affects the function and the interactions of MOR with potential molecular partners (for example G proteins or β-arrestin) A method of siRNA interference was used to knock down the following selected signaling molecules: Gαi1, Gαi2, Gαi3, Gαz and β-arrestin2. The effect of biased ligands on lateral mobility of MOR in the plasma membrane and on activity of adenylyl cyclase (AC) was examined under these conditions. We observed a possible involvement of Gαz subunit in the lateral mobility of MOR after the effect of morphine and endomorphin-2. The lateral mobility of MOR was significantly increased in cells lacking Gαi2 or Gαi3 or β-arrestin2. In this case the MOR was in inactive state....
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Studies on molecular interactions of the mu-opioid and TRPV1 receptors
Melkes, Barbora ; Novotný, Jiří (advisor) ; Blahoš, Jaroslav (referee) ; Krůšek, Jan (referee)
In this work, we investigated the behavior of the -opioid receptor (MOR) and the transient receptor potential vanilloid 1 (TRPV1) ion channel in the plasma membrane and their mutual communication. Both these receptors are implicated in pain perception and analgesia. We observed that the lateral mobility of MOR was strongly affected by different biased opioid agonists. DAMGO and endomorphin-2 display opposite bias towards MOR. According to our results, they also have the opposite effects on the mobility of MOR. Morphine induced only small changes in the mobility of MOR. Moreover, cholesterol depletion and blockage of G protein signaling by pertussis toxin (PTX) affected the ability of different MOR agonists to alter MOR mobility in a unique manner. The effects of DAMGO and endomorphin-2 were compromised under these conditions. On the other hand, we observed increased movement of MOR after the addition of morphine. PTX alone did not affect receptor movement, but it completely disrupted the effect of cholesterol depletion on morphine induced changes the mobility of MOR. Next we studied the mobility of TRPV1. The TRPV1 agonist capsaicin changed the lateral mobility of TRPV1. Surprisingly, after adding the MOR antagonist naloxone, the apparent diffusion coefficient of TRPV1 but to a lower extent than...
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A study of molecular interactions of the μ-opioid receptor: the effect of biased ligands
Marková, Vendula ; Novotný, Jiří (advisor) ; Rudajev, Vladimír (referee)
G protein-coupled receptors (GPCRs) are the largest group of membrane-bound receptors. Transmission of signals into the cell interior is mediated through the interactions of these receptors with other signaling molecules. Nowadays, a great attention is devoted to biased ligands which are able to alter the conformation of the receptor in a specific way and thus distinctly affect its function. This diploma thesis was focused on a study of µ-opioid receptor (MOR), which is important in nociception. The aim of this study was to find out, how the activation of MOR by specific biased ligands (morphine, endomorphin-2 and DAMGO) affects the function and the interactions of MOR with potential molecular partners (for example G proteins or β-arrestin) A method of siRNA interference was used to knock down the following selected signaling molecules: Gαi1, Gαi2, Gαi3, Gαz and β-arrestin2. The effect of biased ligands on lateral mobility of MOR in the plasma membrane and on activity of adenylyl cyclase (AC) was examined under these conditions. We observed a possible involvement of Gαz subunit in the lateral mobility of MOR after the effect of morphine and endomorphin-2. The lateral mobility of MOR was significantly increased in cells lacking Gαi2 or Gαi3 or β-arrestin2. In this case the MOR was in inactive state....
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Influence of protein SGIP1 on partners participating in signalization of cannabinoid receptor 1
Pejšková, Lucie ; Blahoš, Jaroslav (advisor) ; Novotný, Jiří (referee)
The G-protein-coupled receptor (GPCR) family represents the largest family of cell surface receptors. GPCRs are activated by endogenous or exogenous ligands, and are targets for more than a quarter of currently used drugs. Activation of receptors initiates intracellular signaling pathways. This way the membrane receptors transfer information from the outside environment into the cell. Based on the signal the cell can respond to the changes of the environment. Key observation important for this thesis is interplay of cannabinoid and opioid signaling in vivo, which can have significant physiological effects1 . Cannabinoid receptor 1 (CB1R) and µ opioid receptor (MOR) belong to the rhodopsin family of receptors, and both are coupled with Gαi/o proteins2 . Both are located in certain areas in central nervous system (CNS) and share a lot of important features. Activation of both of the receptors leads to inhibition of adenylyl cyclase, thus decreasing the level of cyclic adenosine monophosphate in the cell, and modulates extracellular regulated kinase 1 and 2 (ERK1/2)2 . In view of the numerous anatomical, biochemical and pharmacological evidence supporting the existence of the functional interaction between opioid3 and cannabinoid receptor systems this topic became interesting for our research. In our...
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The role of spinal TRPV1 receptors in nociceptive signalling and the modulatory effect of chemokine CCL2 and µ-opioid receptor agonists
Šulcová, Dominika ; Paleček, Jiří (advisor) ; Krůšek, Jan (referee)
The first nociceptive synapse in the spinal cord dorsal horn represents an important site, where nociceptive synaptic transmission can be modulated under pathological conditions. One of the modulatory mechanism involves activation of the transient receptor potential vanilloid 1 (TRPV1) that is expressed on central terminals of primary nociceptive neurons, where it regulates release of neurotransmitters and neuromodulators. Previous studies suggested that changes in TRPV1 activity may be related to effects of chemokine CCL2 (C-C motif ligand 2) and may be also involved in synaptic transmission modulation after µ-opioid receptors (MOP-R) activation. Because CCL2 receptors CCR2 often co-localize with TRPV1 and MOP-R, the goal of this work was to studypossible interactions of these receptors on the pre-synaptic endings of primaryafferents in the spinal cord dorsal horn and their role in nociceptive signalling under pathological conditions. The presented thesis focused on the effect of CCL2 during peripheral neuropathy and its interference with µ-opioid receptor activation. To studysynaptic transmission at the spinal cord level, patch-clamp recordings of excitatory post-synaptic currents (EPSC) in superficial spinal cord dorsal horn neurons in acute lumbar spinal cord slices from rats was used....
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