GABAB receptors (GBRs) are G protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the brain. GBRs regulate fast synaptic transmission by gating Ca2+ and K+ channels via the Gβγ subunits of the activated G protein. It has been demonstrated that auxiliary GBR subunits, the KCTD proteins, shorten onset and rise time and increase desensitization of receptor-induced K+ currents. KCTD proteins increase desensitization of K+ currents by scavenging Gβγ from the channel, yet the mechanism responsible for the rapid activation of K+ currents has remained elusive. In this study, we demonstrate that KCTD proteins preassemble Gβγ at GBRs. The preassembly obviates the need for diffusion-limited G protein recruitment to the receptor, thereby accelerating G protein activation and, as a result, K+ channel activation. Preassembly of Gβγ at the receptor relies on the interaction of KCTD proteins with a loop protruding from the seven-bladed propeller of Gβ subunits. The binding site is shared between Gβ1 and Gβ2, limiting the interaction of KCTD proteins to these particular Gβ isoforms. Substituting residues in the KCTD binding site of Gβ1 with those from Gβ3 hinders the preassembly of Gβγ with GBRs, delays onset and prolongs rise time of receptor-activated K+ currents. The KCTD-Gβ interface, therefore, represents a target for pharmacological modulation of channel gating by GBRs.

• MeSH
• draslíkové kanály metabolismus   genetika   MeSH
• gating iontového kanálu * fyziologie   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• proteiny vázající GTP - beta-podjednotky * metabolismus   genetika   MeSH
• proteiny vázající GTP - gama-podjednotky * metabolismus   genetika   MeSH
• receptory GABA-B * metabolismus   genetika   MeSH
• Xenopus laevis MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Genetic causes of canine mast cell tumours (MCTs), except for mutations in the KIT gene detected in some MCTs, are generally unknown. We used whole exome sequencing to reveal mutation spectra in canine MCTs. We detected somatic mutations in 87 genes including 10 genes recognized as human cancer drivers. Besides KIT, 14 other genes were recurrently mutated. Subsequently, we performed next generation sequencing of a panel of 50 selected genes in additional MCT samples. In this group, the most frequently altered gene was GNB1 showing a recurrent dinucleotide substitution at position of Gly116 in 30% of the MCT samples (n = 6/20) and Ile80 substitution accompanied by a splice region mutation in one case. We extended the study by analysis of the above mentioned GNB1 regions in additional MCT samples by Sanger sequencing, and assessed the overall prevalence of GNB1 mutations to 17.3% (n = 14/81), which is similar to the prevalence of KIT alterations. Our results indicate that GNB1 mutations are probably involved in canine MCT pathogenesis in both cutaneous and subcutaneous MCT cases. As opposed to KIT alterations, the presence of GNB1 mutations did not negatively affect survival times, and our data even showed a trend towards positive prognosis. If our results are confirmed in a larger number of MCTs, an extension of molecular testing of canine MCTs by GNB1 analysis would help to refine the molecular stratification of MCTs, and become useful for targeted treatment strategies.

• MeSH
• mastocytární sarkom genetika   patologie   veterinární   MeSH
• mastocyty patologie   MeSH
• mutace MeSH
• nemoci psů genetika   patologie   MeSH
• proteiny vázající GTP - beta-podjednotky genetika   MeSH
• protoonkogenní proteiny c-kit genetika   MeSH
• psi MeSH
• vysoce účinné nukleotidové sekvenování veterinární   MeSH
• zvířata MeSH
• Check Tag
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The association of GNB4 with Charcot-Marie-Tooth (CMT) has recently been described in a publication by Soong et al. (Soong, et al., 2013). Here we present a patient with CMT in whom whole exome sequencing identified the mutation p.Lys57Glu in the GNB4 gene (NM_021629.3:c.169A>G). The patient, now 41 years old, is a sporadic case in the family. At the age of 35 he presented with severe disability (CMT neuropathy score 29), profound muscle atrophies, pes cavus and scoliosis. Previously, the patient was tested for PMP22 duplications/deletions and later also with 64 CMT gene panel, with no causal variant found. Subsequently, whole exome sequencing was performed. The p.Lys57Glu in the GNB4 gene was identified as the most probable causal variant, the mutation is not present in the patient's parents, neither in his unaffected sister, therefore we assume that the mutation arose de novo. Taken together, these findings support the causal and pathogenic character of the variant. Our report provides important evidence that GNB4 should become an established CMT gene and our findings confirm the original publication by Soong et al. (2013).

• MeSH
• Charcotova-Marieova-Toothova nemoc genetika   patologie   patofyziologie   MeSH
• dospělí MeSH
• lidé MeSH
• mutace MeSH
• proteiny vázající GTP - beta-podjednotky genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• Geografické názvy
• Česká republika MeSH

Leukemias harboring the ETV6-ABL1 fusion represent a rare subset of hematological malignancies with unfavorable outcomes. The constitutively active chimeric Etv6-Abl1 tyrosine kinase can be specifically inhibited by tyrosine kinase inhibitors (TKIs). Although TKIs represent an important therapeutic tool, so far, the mechanism underlying the potential TKI resistance in ETV6-ABL1-positive malignancies has not been studied in detail. To address this issue, we established a TKI-resistant ETV6-ABL1-positive leukemic cell line through long-term exposure to imatinib. ETV6-ABL1-dependent mechanisms (including fusion gene/protein mutation, amplification, enhanced expression or phosphorylation) and increased TKI efflux were excluded as potential causes of resistance. We showed that TKI effectively inhibited the Etv6-Abl1 kinase activity in resistant cells, and using short hairpin RNA (shRNA)-mediated silencing, we confirmed that the resistant cells became independent from the ETV6-ABL1 oncogene. Through analysis of the genomic and proteomic profiles of resistant cells, we identified an acquired mutation in the GNB1 gene, K89M, as the most likely cause of the resistance. We showed that cells harboring mutated GNB1 were capable of restoring signaling through the phosphoinositide-3-kinase (PI3K)/Akt/mTOR and mitogen-activated protein kinase (MAPK) pathways, whose activation is inhibited by TKI. This alternative GNB1K89M-mediated pro-survival signaling rendered ETV6-ABL1-positive leukemic cells resistant to TKI therapy. The mechanism of TKI resistance is independent of the targeted chimeric kinase and thus is potentially relevant not only to ETV6-ABL1-positive leukemias but also to a wider spectrum of malignancies treated by kinase inhibitors.

• MeSH
• chemorezistence účinky léků   MeSH
• fúzní onkogenní proteiny genetika   MeSH
• imatinib mesylát aplikace a dávkování   MeSH
• inhibitory proteinkinas aplikace a dávkování   MeSH
• leukemie farmakoterapie   genetika   patologie   MeSH
• lidé MeSH
• malá interferující RNA genetika   MeSH
• mutace MeSH
• nádorové buněčné linie MeSH
• proteiny vázající GTP - beta-podjednotky genetika   MeSH
• signální transdukce účinky léků   MeSH
• tyrosinkinasy genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Neuronal voltage-gated calcium channels have evolved as one of the most important players for calcium entry into presynaptic endings responsible for the release of neurotransmitters. In turn, and to fine-tune synaptic activity and neuronal communication, numerous neurotransmitters exert a potent negative feedback over the calcium signal provided by G protein-coupled receptors. This regulation pathway of physiologic importance is also extensively exploited for therapeutic purposes, for instance in the treatment of neuropathic pain by morphine and other μ-opioid receptor agonists. However, despite more than three decades of intensive research, important questions remain unsolved regarding the molecular and cellular mechanisms of direct G protein inhibition of voltage-gated calcium channels. In this study, we revisit this particular regulation and explore new considerations.

• MeSH
• blokátory kalciových kanálů farmakologie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• neurony metabolismus   MeSH
• presynaptické terminály metabolismus   MeSH
• proteiny vázající GTP - beta-podjednotky metabolismus   MeSH
• proteiny vázající GTP - gama-podjednotky metabolismus   MeSH
• proteiny vázající GTP metabolismus   MeSH
• receptory spřažené s G-proteiny metabolismus   MeSH
• vápníkové kanály chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Activation of K(+) channels by the G protein βγ subunits is an important signaling mechanism of G-protein-coupled receptors. Typically, receptor-activated K(+) currents desensitize in the sustained presence of agonists to avoid excessive effects on cellular activity. The auxiliary GABAB receptor subunit KCTD12 induces fast and pronounced desensitization of the K(+) current response. Using proteomic and electrophysiological approaches, we now show that KCTD12-induced desensitization results from a dual interaction with the G protein: constitutive binding stabilizes the heterotrimeric G protein at the receptor, whereas dynamic binding to the receptor-activated Gβγ subunits induces desensitization by uncoupling Gβγ from the effector K(+) channel. While receptor-free KCTD12 desensitizes K(+) currents activated by other GPCRs in vitro, native KCTD12 is exclusively associated with GABAB receptors. Accordingly, genetic ablation of KCTD12 specifically alters GABAB responses in the brain. Our results show that GABAB receptors are endowed with fast and reversible desensitization by harnessing KCTD12 that intercepts Gβγ signaling.

• MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• dovnitř usměrňující draslíkové kanály spřažené s G proteiny metabolismus   MeSH
• HEK293 buňky MeSH
• intracelulární signální peptidy a proteiny metabolismus   MeSH
• lidé MeSH
• mozek metabolismus   MeSH
• myši MeSH
• proteiny vázající GTP - beta-podjednotky metabolismus   MeSH
• proteiny vázající GTP - gama-podjednotky metabolismus   MeSH
• receptory GABA-B chemie   metabolismus   MeSH
• receptory GABA metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Although most heterotrimeric G proteins are thought to dissociate into Gα and Gβγ subunits upon activation, the evidence in the Gi/o family has long been inconsistent and contradictory. The Gi/o protein family mediates inhibition of cAMP production and regulates the activity of ion channels. On the basis of experimental evidence, both heterotrimer dissociation and rearrangement have been postulated as crucial steps of Gi/o protein activation and signal transduction. We have now investigated the process of Gi/o activation in living cells directly by two-photon polarization microscopy and indirectly by observations of G protein-coupled receptor kinase-derived polypeptides. Our observations of existing fluorescently labeled and non-modified Gαi/o constructs indicate that the molecular mechanism of Gαi/o activation is affected by the presence and localization of the fluorescent label. All investigated non-labeled, non-modified Gi/o complexes dissociate extensively upon activation. The dissociated subunits can activate downstream effectors and are thus likely to be the major activated Gi/o form. Constructs of Gαi/o subunits fluorescently labeled at the N terminus (GAP43-CFP-Gαi/o) seem to faithfully reproduce the behavior of the non-modified Gαi/o subunits. Gαi constructs labeled within the helical domain (Gαi-L91-YFP) largely do not dissociate upon activation, yet still activate downstream effectors, suggesting that the dissociation seen in non-modified Gαi/o proteins is not required for downstream signaling. Our results appear to reconcile disparate published data and settle a long running dispute.

• MeSH
• aktivace enzymů fyziologie   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• protein GAP-43 genetika   metabolismus   MeSH
• proteiny vázající GTP - alfa-podjednotky Gi-Go genetika   metabolismus   MeSH
• proteiny vázající GTP - beta-podjednotky genetika   metabolismus   MeSH
• proteiny vázající GTP - gama-podjednotky genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

With the aim to understand the onset of expression and developmental profile of plasma membrane (PM) content /density of crucial components of GABA(B)-R signaling cascade, GABA(B)-R1a, GABA(B)-R1b, GABA(B)-R2, G(i)1/G(i)2alpha, G(i)3alpha, G(o)alpha, G(z)alpha and Gbeta subunit proteins were determined by quantitative immunoblotting and compared in PM isolated from brain cortex of rats of different ages: between postnatal-day-1 (PD1) and 90 (PD90). PM density of GABA(B)-R1a, GABA(B)-R2, G(i)1/G(i)2alpha, G(i)3alpha, G(o)alpha, G(z)alpha and Gbeta was high already at birth and further development was reflected in parallel decrease of both GABA(B)-R1a and GABA(B)-R2 subunits. The major decrease of GABA(B)-R1a and GABA(B)-R2 occurred between the birth and PD15: to 55 % (R1a, **) and 51 % (R2, **), respectively. Contrarily, PM level of the cognate G-proteins G(i)1/G(i)2alpha, G(i)3alpha, G(o)alpha, G(z)alpha and Gbeta was unchanged in the course of the whole postnatal period of brain cortex development. Maturation of GABA(B)-R cascade was substantially different from ontogenetic profile of prototypical plasma membrane marker, Na, K-ATPase, which was low at birth and further development was reflected in continuous increase of PM density of this enzyme. Major change occurred between the birth and PD25. In adult rats, membrane content of Na, K-ATPase was 3-times higher than around the birth.

• MeSH
• buněčná membrána metabolismus   MeSH
• heterotrimerní G-proteiny metabolismus   MeSH
• krysa rodu rattus MeSH
• mozková kůra růst a vývoj   metabolismus   MeSH
• proteiny vázající GTP - alfa-podjednotka Gi2 metabolismus   MeSH
• proteiny vázající GTP - alfa-podjednotky Gi-Go metabolismus   MeSH
• proteiny vázající GTP - alfa-podjednotky metabolismus   MeSH
• proteiny vázající GTP - beta-podjednotky metabolismus   MeSH
• receptory GABA-B metabolismus   MeSH
• sodíko-draslíková ATPasa metabolismus   MeSH
• věkové faktory MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The aim of the project is the proof of relation between presence of arterial hypertension and progression of selected chronic renal diseases into the end stage renal failure and some gene polymorphisms supposed to be included in pathogenesis.

Cílem projektu je prokázat vztah mezi přítomností arteriální hypertenze (AH) a progresí vybraných chronických chorob ledvin do do stadia chronického selhání ledvin (CHSL) a některými polymorfismy genů, které se mohou v patogenezi AH/CHSL podílet.

• MeSH
• endoteliální buňky MeSH
• IgA nefropatie MeSH
• lékařská genetika MeSH
• polycystická choroba ledvin etiologie   MeSH
• polymorfismus genetický MeSH
• proteiny vázající GTP - beta-podjednotky MeSH
• renální insuficience MeSH
• renovaskulární hypertenze MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• nefrologie
• biologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR