Mechanisms of Transforming Growth Factor-β Receptor Endocytosis and Intracellular Sorting Differ between Fibroblasts and Epithelial Cells

Transforming growth factor-βs (TGF-β) are multifunctional proteins capable of either stimulating or inhibiting mitosis, depending on the cell type. These diverse cellular responses are caused by stimulating a single receptor complex composed of type I and type II receptors. Using a chimeric receptor model where the granulocyte/monocyte colony-stimulating factor receptor ligand binding domains are fused to the transmembrane and cytoplasmic signaling domains of the TGF-β type I and II receptors, we wished to describe the role(s) of specific amino acid residues in regulating ligand-mediated endocytosis and signaling in fibroblasts and epithelial cells. Specific point mutations were introduced at Y182, T200, and Y249 of the type I receptor and K277 and P525 of the type II receptor. Mutation of either Y182 or Y249, residues within two putative consensus tyrosine-based internalization motifs, had no effect on endocytosis or signaling. This is in contrast to mutation of T200 to valine, which resulted in ablation of signaling in both cell types, while only abolishing receptor down-regulation in fibroblasts. Moreover, in the absence of ligand, both fibroblasts and epithelial cells constitutively internalize and recycle the TGF-β receptor complex back to the plasma membrane. The data indicate fundamental differences between mesenchymal and epithelial cells in endocytic sorting and suggest that ligand binding diverts heteromeric receptors from the default recycling pool to a pathway mediating receptor down-regulation and signaling.

Crystallographic structures of the ligand-binding domains of the androgen receptor and its T877A mutant complexed with the natural agonist dihydrotestosterone

The structures of the ligand-binding domains (LBD) of the wild-type androgen receptor (AR) and the T877A mutant corresponding to that in LNCaP cells, both bound to dihydrotestosterone, have been refined at 2.0 Å resolution. In contrast to the homodimer seen in the retinoid-X receptor and estrogen receptor LBD structures, the AR LBD is monomeric, possibly because of the extended C terminus of AR, which lies in a groove at the dimerization interface. Binding of the natural ligand dihydrotestosterone by the mutant LBD involves interactions with the same residues as in the wild-type receptor, with the exception of the side chain of threonine 877, which is an alanine residue in the mutant. This structural difference in the binding pocket can explain the ability of the mutant AR found in LNCaP cells (T877A) to accommodate progesterone and other ligands that the wild-type receptor cannot.

Defective STAT signaling by the leptin receptor in diabetic mice.

Leptin and its receptor, obese receptor (OB-R), comprise an important signaling system for the regulation of body weight. Splice variants of OB-R mRNA encode proteins that differ in the length of their cytoplasmic domains. We cloned a long isoform of the wild-type leptin receptor that is preferentially expressed in the hypothalamus and show that it can activate signal transducers and activators of transcription (STAT)-3, STAT-5, and STAT-6. A point mutation within the OB-R gene of diabetic (db) mice generates a new splice donor site that dramatically reduces expression of this long isoform in homozygous db/db mice. In contrast, an OB-R protein with a shorter cytoplasmic domain is present in both db/db and wild-type mice. We show that this short isoform is unable to activate the STAT pathway. These data provide further evidence that the mutation in OB-R causes the db/db phenotype and identify three STAT proteins as potential mediators of the anti-obesity effects of leptin.

Palmitoylation of the GluR6 kainate receptor.

The G-protein-coupled metabotropic glutamate receptor mGluR1 alpha and the ionotropic glutamate receptor GluR6 were examined for posttranslational palmitoylation. Recombinant receptors were expressed in baculovirus-infected insect cells or in human embryonic kidney cells and were metabolically labeled with [3H]palmitic acid. The metabotropic mGluR1 alpha receptor was not labeled whereas the GluR6 kainate receptor was labeled after incubation with [3H]palmitate. The [3H]palmitate labeling of GluR6 was eliminated by treatment with hydroxylamine, indicating that the labeling was due to palmitoylation at a cysteine residue via a thioester bond. Site-directed mutagenesis was used to demonstrate that palmitoylation of GluR6 occurs at two cysteine residues, C827 and C840, located in the carboxyl-terminal domain of the molecule. A comparison of the electrophysiological properties of the wild-type and unpalmitoylated mutant receptor (C827A, C840A) showed that the kainate-gated currents produced by the unpalmitoylated mutant receptor were indistinguishable from those of the wild-type GluR6. The unpalmitoylated mutant was a better substrate for protein kinase C than the wild-type GluR6 receptor. These data indicate that palmitoylation may not modulate kainate channel function directly but instead affect function indirectly by regulating the phosphorylation state of the receptor.

Studies on mu and delta opioid receptor selectivity utilizing chimeric and site-mutagenized receptors.

Opioid receptors are members of the guanine nucleotide binding protein (G protein)-coupled receptor family. Three types of opioid receptors have been cloned and characterized and are referred to as the delta, kappa and mu types. Analysis of receptor chimeras and site-directed mutant receptors has provided a great deal of information about functionally important amino acid side chains that constitute the ligand-binding domains and G-protein-coupling domains of G-protein-coupled receptors. We have constructed delta/mu opioid receptor chimeras that were express in human embryonic kidney 293 cells in order to define receptor domains that are responsible for receptor type selectivity. All chimeric receptors and wild-type delta and mu opioid receptors displayed high-affinity binding of etorphine (an agonist), naloxone (an antagonist), and bremazocine (a mixed agonist/antagonist). In contrast, chimeras that lacked the putative first extracellular loop of the mu receptor did not bind the mu-selective peptide [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO). Chimeras that lacked the putative third extracellular loop of the delta receptor did not bind the delta-selective peptide, [D-Ser2,D-Leu5]enkephalin-Thr (DSLET). Point mutations in the putative third extracellular loop of the wild-type delta receptor that converted vicinal arginine residues to glutamine abolished DSLET binding while not affecting bremazocine, etorphine, and naltrindole binding. We conclude that amino acids in the putative first extracellular loop of the mu receptor are critical for high-affinity DAMGO binding and that arginine residues in the putative third extracellular loop of the delta receptor are important for high-affinity DSLET binding.

The preimplantation mouse embryo is a target for cannabinoid ligand-receptor signaling.

Using a reverse transcription-coupled PCR, we demonstrated that both brain and spleen type cannabinoid receptor (CB1-R and CB2-R, respectively) mRNAs are expressed in the preimplantation mouse embryo. The CB1-R mRNA expression was coincident with the activation of the embryonic genome late in the two-cell stage, whereas the CB2-R mRNA was present from the one-cell through the blastocyst stages. The major psychoactive component of marijuana (-)-delta-9-tetrahydrocannabinol [(-)-THC] inhibited forskolin-stimulated cAMP generation in the blastocyst, and this inhibition was prevented by pertussis toxin. However, the inactive cannabinoid cannabidiol (CBD) failed to influence this response. These results suggest that cannabinoid receptors in the embryo are coupled to inhibitory guanine nucleotide binding proteins. Further, the oviduct and uterus exhibited the enzymatic capacity to synthesize the putative endogenous cannabinoid ligand arachidonylethanolamide (anandamide). Synthetic and natural cannabinoid agonists [WIN 55,212-2, CP 55,940, (-)-THC, and anandamide], but not CBD or arachidonic acid, arrested the development of two-cell embryos primarily between the four-cell and eight-cell stages in vitro in a dose-dependent manner. Anandamide also interfered with the development of eight-cell embryos to blastocysts in culture. The autoradiographic studies readily detected binding of [3H]anandamide in embryos at all stages of development. Positive signals were present in one-cell embryos and all blastomeres of two-cell through four-cell embryos. However, most of the binding sites in eight-cell embryos and morulae were present in the outer cells. In the blastocyst, these signals were primarily localized in the mural trophectoderm with low levels of signals in the polar trophectoderm, while little or no signals were noted in inner cell mass cells.These results establish that the preimplantation mouse embryo is a target for cannabinoid ligands. Consequently, many of the adverse effects of cannabinoids observed during pregnancy could be mediated via these cannabinoid receptors. Although the physiological significance of the cannabinoid ligand-receptor signaling in normal preimplantation embryo development is not yet clear, the regulation of embryonic cAMP and/or Ca2+ levels via this signaling pathway may be important for normal embryonic development and/or implantation.

Cannabinoid ligand-receptor signaling in the mouse uterus.

Using RNA (Northern) blot hybridization and reverse transcription-PCR, we demonstrate that the brain-type cannabinoid receptor (CB1-R) mRNA, but not the spleen-type cannabinoid receptor (CB2-R) mRNA, is expressed in the mouse uterus and that this organ has the capacity to synthesize the putative endogenous cannabinoid ligand, anandamide (arachidonylethanolamide). The psychoactive cannabinoid component of marijuana--delta 9-tetrahydrocannabinol (THC)--or anandamide, but not the inactive and nonpsychoactive cannabidiol (CBD), inhibited forskolin-stimulated cyclic AMP formation in the mouse uterus, which was prevented by pertussis toxin pretreatment. These results suggest that uterine CB1-R is coupled to inhibitory guanine nucleotide-binding protein and is biologically active. Autoradiographic studies identified ligand binding sites ([3H]anandamide) in the uterine epithelium and stromal cells, suggesting that these cells are perhaps the targets for cannabinoid action. Scatchard analysis of the binding of [3H]WIN 55212-2, another cannabinoid receptor ligand, showed a single class of high-affinity binding sites in the endometrium with an apparent Kd of 2.4 nM and Bmax of 5.4 x 10(9) molecules per mg of protein. The gene encoding lactoferrin is an estrogen-responsive gene in the mouse uterus that was rapidly and transiently up-regulated by THC, but not by CBD, in ovariectomized mice in the absence of ovarian steroids. This effect, unlike that of 17 beta-estradiol (E2), was not influenced by a pure antiestrogen, ICI 182780, suggesting that the THC-induced uterine lactoferrin gene expression does not involve estrogen receptors. We propose that the uterus is a new target for cannabinoid ligand-receptor signaling.

O antagonismo do receptor de serotonina do tipo 7 da substância cinzenta periaquedutal dorsal reduz a ansiedade experimental basal, mas não aquela gerada pela retirada do etanol em ratos

Ethanol-dependent individuals who reduce or discontinue its use may present Alcohol Withdrawal Syndrome, which is characterized by unpleasant signs and symptoms, such as anxiety, that may trigger relapses. Ethanol, a psychotropic drug, is able to promote behavioral and neurophysiological changes, acting on different neurotransmitter systems, including the serotonergic, which has also been directly associated with aversive states, including anxiety. This study aimed to investigate the participation of type 7 serotonin receptor (5-HT7) of the dorsal periaqueductal gray (DPAG) on basal experimental anxiety and that caused by ethanol withdrawal. For this, 75-100 days old Wistar rats were subjected to two experiments. On the first one, animals underwent stereotactic surgery for implantation of guide cannulas used for administration of the drug directly into the DPAG. After seven days, the animals received doses of 2.5; 5 and 10 nmols of type 7 receptor antagonist SB269970 (SB) or vehicle intra-DPAG and, ten minutes after, they were exposed to elevated plus maze (EPM). In the following day, the animals were submitted to the same treatment and tested in the open field (OF). In the second experiment, animals received increasing concentrations (2%, 4%, 6%) of ethanol as the only source of liquid diet or water (control group), both with free access to chow. Seventy two hours and ninety six hours after the ethanol withdrawal, animals received SB (2.5 and 5.0 nmols) intraDPAG ten minutes before the test in the LCE and OF, respectively. In experiment 1, the dose of antagonist 10 nmols was able of reversing the anxiety generated by EPM. In the experiment 2, ineffective SB doses on the LCE (2.5 and 5.0 nmol) were not able to reverse the anxiety caused by the ethanol withdrawal in the EPM, although the dose of 2.5 nmols of SB has reversed its hipolocomotor effect in this test. This result suggests that the 5-HT7 receptor is involved in the modulation of the basal experimental anxiety in rats, but not in the anxiety caused by ethanol withdrawal in the DPAG.

Angiotensin receptor I stimulates osteoprogenitor proliferation through TGFβ-mediated signaling

Clinical studies of large human populations and pharmacological interventions in rodent models have recently suggested that anti-hypertensive drugs that target angiotensin II (Ang II) activity may also improve loss of bone mineral density. Here we identified in a genetic screen the Ang II type I receptor (AT1R) as a potential determinant of osteogenic differentiation and, implicitly, bone formation. Silencing of AT1R expression by RNA interference severely impaired the maturation of a multipotent mesenchymal cell line (W20-17) along the osteoblastic lineage. The same effect was also observed after the addition of the AT1R antagonist losartan but not the AT2R inhibitor PD123,319. Additional cell culture assays traced the time of greatest losartan action to the early stages of W20-17 differentiation, namely during cell proliferation. Indeed, addition of Ang II increased proliferation of differentiating W20-17 and primary mesenchymal stem cells and this stimulation was reversed by losartan treatment. Cells treated with losartan also displayed an appreciable decrease of activated (phosphorylated)-Smad2/3 proteins. Moreover, Ang II treatment elevated endogenous transforming growth factor β (TGFβ) expression considerably and in an AT1R-dependent manner. Finally, exogenous TGFβ was able to restore high proliferative activity to W20-17 cells that were treated with both Ang II and losartan. Collectively, these results suggest a novel mechanism of Ang II action in bone metabolism that is mediated by TGFβ and targets proliferation of osteoblast progenitors.

Fluorine-18-labeled estrogens, progestins and corticosteroids for receptor-based imaging of breast tumors and target areas of the brain

Estrogens can be labeled with the positron-emitting radionuclide fluorine-18 (t$\sb{1/2}$ = 110 min) by fluoride ion (n-Bu$\sb4$N$\sp{18}$F) displacement of a 16$\beta$-trifluoromethanesulfonate (triflate) derivative of the corresponding estrone 3-triflate, and purification by HPLC. That sequence has been used to synthesize the 11$\beta$-methoxy 1 and 11$\beta$-ethyl 2 analogues of the breast tumor imaging agent, 16$\alpha$-($\sp{18}$F) fluoro-17$\beta$-estradiol (FES). Tissue distribution studies of 1 and 2 in immature female rats show high selectivity for target tissue (T, uterus) vs non-target (NT, muscle and lung), with T/NT ratios being 43 and 17 at one hour after injection for 1 and 2, respectively. The parent estrogen FES has previously been shown to display an intermediate value for tissue selectivity.

Prognostic significance of IGF and ECM induced signalling proteins in breast cancer patients

Breast cancer is a leading contributor to the burden of disease in Australia. Fortunately, the recent introduction of diverse therapeutic strategies have improved the survival outcome for many women. Despite this, the clinical management of breast cancer remains problematic as not all approaches are sufficiently sophisticated to take into account the heterogeneity of this disease and are unable to predict disease progression, in particular, metastasis. As such, women with good prognostic outcomes are exposed to the side effects of therapies without added benefit. Furthermore, women with aggressive disease for whom these advanced treatments would deliver benefit cannot be distinguished and opportunities for more intensive or novel treatment are lost. This study is designed to identify novel factors associated with disease progression, and the potential to inform disease prognosis. Frequently overlooked, yet common mediators of disease are the interactions that take place between the insulin-like growth factor (IGF) system and the extracellular matrix (ECM). Our laboratory has previously demonstrated that multiprotein insulin-like growth factor-I (IGF-I): insulin-like growth factor binding protein (IGFBP): vitronectin (VN) complexes stimulate migration of breast cancer cells in vitro, via the cooperative involvement of the insulin-like growth factor type I receptor (IGF-IR) and VN-binding integrins. However, the effects of IGF and ECM protein interactions on the dissemination and progression of breast cancer in vivo are unknown. It was hypothesised that interactions between proteins required for IGF induced signalling events and those within the ECM contribute to breast cancer metastasis and are prognostic and predictive indicators of patient outcome. To address this hypothesis, semiquantitative immunohistochemistry (IHC) analyses were performed to compare the extracellular and subcellular distribution of IGF and ECM induced signalling proteins between matched normal, primary cancer, and metastatic cancer among archival formalin-fixed paraffin-embedded (FFPE) breast tissue samples collected from women attending the Princess Alexandra Hospital, Brisbane. Multivariate Cox proportional hazards (PH) regression survival models in conjunction with a modified „purposeful selection of covariates. method were applied to determine the prognostic potential of these proteins. This study provides the first in-depth, compartmentalised analysis of the distribution of IGF and ECM induced signalling proteins. As protein function and protein localisation are closely correlated, these findings provide novel insights into IGF signalling and ECM protein function during breast cancer development and progression. Distinct IGF signalling and ECM protein immunoreactivity was observed in the stroma and/or in subcellular locations in normal breast, primary cancer and metastatic cancer tissues. Analysis of the presence and location of stratifin (SFN) suggested a causal relationship in ECM remodelling events during breast cancer development and progression. The results of this study have also suggested that fibronectin (FN) and ¥â1 integrin are important for the formation of invadopodia and epithelial-to-mesenchymal transition (EMT) events. Our data also highlighted the importance of the temporal and spatial distribution of IGF induced signalling proteins in breast cancer metastasis; in particular, SFN, enhancer-of-split and hairy-related protein 2 (SHARP-2), total-akt/protein kinase B 1 (Total-AKT1), phosphorylated-akt/protein kinase B (P-AKT), extracellular signal-related kinase-1 and extracellular signal-related kinase-2 (ERK1/2) and phosphorylated-extracellular signal-related kinase-1 and extracellular signal-related kinase-2 (P-ERK1/2). Multivariate survival models were created from the immunohistochemical data. These models were found to fit well with these data with very high statistical confidence. Numerous prognostic confounding effects and effect modifications were identified among elements of the ECM and IGF signalling cascade and corroborate the survival models. This finding provides further evidence for the prognostic potential of IGF and ECM induced signalling proteins. In addition, the adjusted measures of associations obtained in this study have strengthened the validity and utility of the resulting models. The findings from this study provide insights into the biological interactions that occur during the development of breast tissue and contribute to disease progression. Importantly, these multivariate survival models could provide important prognostic and predictive indicators that assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy. The outcomes of this study further inform the development of new therapeutics to aid patient recovery. The findings from this study have widespread clinical application in the diagnosis of disease and prognosis of disease progression, and inform the most appropriate clinical management of individuals with breast cancer.

Differential expression profiling of components associated with exoskeletal hardening in crustaceans

Background: Exoskeletal hardening in crustaceans can be attributed to mineralization and sclerotization of the organic matrix. Glycoproteins have been implicated in the calcification process of many matrices. Sclerotization, on the other hand, is catalysed by phenoloxidases, which also play a role in melanization and the immunological response in arthropods. Custom cDNA microarrays from Portunus pelagicus were used to identify genes possibly associated with the activation pathways involved in these processes. Results: Two genes potentially involved in the recognition of glycosylation, the C-type lectin receptor and the mannose-binding protein, were found to display molt cycle-related differential expression profiles. C-type lectin receptor up-regulation was found to coincide with periods associated with new uncalcified cuticle formation, while the up-regulation of mannose-binding protein occurred only in the post-molt stage, during which calcification takes place, implicating both in the regulation of calcification. Genes presumed to be involved in the phenoloxidase activation pathway that facilitates sclerotization also displayed molt cycle-related differential expression profiles. Members of the serine protease superfamily, trypsin-like and chymotrypsin-like, were up-regulated in the intermolt stage when compared to post-molt, while trypsin-like was also up-regulated in pre-molt compared to ecdysis. Additionally, up-regulation in pre- and intermolt stages was observed by transcripts encoding other phenoloxidase activators including the putative antibacterial protein carcinin-like, and clotting protein precursor-like. Furthermore, hemocyanin, itself with phenoloxidase activity, displayed an identical expression pattern to that of the phenoloxidase activators, i.e. up-regulation in pre- and intermolt. Conclusion: Cuticle hardening in crustaceans is a complex process that is precisely timed to occur in the post-molt stage of the molt cycle. We have identified differential expression patterns of several genes that are believed to be involved in biomineralization and sclerotization and propose possible regulatory mechanisms for these processes based on their expression profiles, such as the potential involvement of C-type lectin receptors and mannose binding protein in the regulation of calcification.

Growth differentiation factor 9 signalling in the ovary

In the ovary, two new members of the large TGF-beta superfamily of growth factors were discovered in the 1990s. The oocyte was shown to express two closely related growth factors that were named growth differentiation factor 9 (GDF-9) and growth differentiation factor 9B (GDF-9B). Both of these proteins are required for normal ovarian follicle development although their individual significance varies between species. GDF-9 and GDF-9B mRNAs are expressed in the human oocytes from the primary follicle stage onwards. This thesis project was aimed to define the signalling mechanisms utilized by the oocyte secreted GDF-9. We used primary cultures of human granulosa luteal cells (hGL) as our cell model, and recombinant adenovirus-mediated gene transfer in manipulating the TGF-b family signalling cascade molecules in these cells. Overexpression of the constitutively active forms of the seven type I receptors, the activin receptor-like kinases 1-7 (ALK1-7), using recombinant adenoviruses caused a specific activation of either the Smad1 or Smad2 pathway proteins depending on the ALK used. Activation of both Smad1 and Smad2 proteins also stimulated the expression of dimeric inhibin B protein in hGL cells. Treatment with recombinant GDF-9 protein induced the specific activation of the Smad2 pathway and stimulated the expression of inhibin betaB subunit mRNA as well as inhibin B protein secretion in our cell model. Recombinant GDF-9 also activated the Smad3-responsive CAGA-luciferase reported construct, and the GDF-9 response in hGL cells was markedly potentiated upon the overexpression of Alk5 by adenoviral gene transduction. Alk5 overexpression also enhanced the GDF-9 induced inhibin B secretion by these cells. Similarly, in a mouse teratocarcinoma cell line P19, GDF-9 could activate the Smad2/3 pathway, and overexpression of ALK5 in COS7 cells rendered them responsive to GDF-9. Furthermore, transfection of rat granulosa cells with small interfering RNA for ALK5 or overexpression of the inhibitory Smad7 resulted in dose-dependent suppression of GDF-9 effects. In conclusion, this thesis shows that both Smad1 and Smad2 pathways are involved in controlling the regulation of inhibin B secretion. Therefore, in addition to endocrine control of inhibin production by the pituitary gonadotropins, also local paracrine factors within in the ovary, like the oocyte-derived growth factors, may contribute to controlling inhibin secretion. This thesis shows as well that like other TGF-beta family ligands, also GDF-9 signalling is mediated by the canonical type I and type II receptors with serine/threonine kinase activity, and the intracellular transcription factors, the Smads. Although GDF-9 binds to the BMP type II receptor, its downstream actions are specifically mediated by the type I receptor, ALK5, and the Smad2 and Smad3 proteins.

Search For Susceptibility Genes In Schizophrenia

Schizophrenia is a severe mental disorder affecting 0.4-1% of the population worldwide. It is characterized by impairments in the perception of reality and by significant social or occupational dysfunction. The disorder is one of the major contributors to the global burden of diseases. Studies of twins, families, and adopted children point to strong genetic components for schizophrenia, but environmental factors also play a role in the pathogenesis of disease. Molecular genetic studies have identified several potential positional candidate genes. The strongest evidence for putative schizophrenia susceptibility loci relates to the genes encoding dysbindin (DTNBP1) and neuregulin (NRG1), but studies lack impressive consistency in the precise genetic regions and alleles implicated. We have studied the role of three potential candidate genes by genotyping 28 single nucleotide polymorphisms in the DNTBP1, NRG1, and AKT1 genes in a large schizophrenia family sample consisting of 441 families with 865 affected individuals from Finland. Our results do not support a major role for these genes in the pathogenesis of schizophrenia in Finland. We have previously identified a region on chromosome 5q21-34 as a susceptibility locus for schizophrenia in a Finnish family sample. Recently, two studies reported association between the γ-aminobutyric acid type A receptor cluster of genes in this region and one study showed suggestive evidence for association with another regional gene encoding clathrin interactor 1 (CLINT1, also called Epsin 4 and ENTH). To further address the significance of these genes under the linkage peak in the Finnish families, we genotyped SNPs of these genes, and observed statistically significant association of variants between GABRG2 and schizophrenia. Furthermore, these variants also seem to affect the functioning of the working memory. Fetal events and obstetric complications are associated with schizophrenia. Rh incompatibility has been implicated as a risk factor for schizophrenia in several epidemiological studies. We conducted a family-based candidate-gene study that assessed the role of maternal-fetal genotype incompatibility at the RhD locus in schizophrenia. There was significant evidence for an RhD maternal-fetal genotype incompatibility, and the risk ratio was estimated at 2.3. This is the first candidate-gene study to explicitly test for and provide evidence of a maternal-fetal genotype incompatibility mechanism in schizophrenia. In conclusion, in this thesis we found evidence that one GABA receptor subunit, GABRG2, is significantly associated with schizophrenia. Furthermore, it also seems to affect to the functioning of the working memory. In addition, an RhD maternal-fetal genotype incompatibility increases the risk of schizophrenia by two-fold.

Nikotiinireseptoreiden kautta vaikuttavat lääkeaineet nikotiiniriippuvuuden ja neurologisten sairauksien hoidossa

Neuronaaliset nikotiinireseptorit liittyvät tupakkariippuvuuden lisäksi moniin neurologisiin sairauksiin, kuten Alzheimerin tautiin, skitsofreniaan, masennukseen ja tarkkaavaisuus- ja ylivilkkaushäiriöön. Nikotiinireseptorien stimulaation on tutkimuksissa havaittu parantavan kognitiota. Useat lääkeyritykset tutkivat nikotiinireseptoriagonisteja ja -antagonisteja eri neurologisten sairauksien hoidossa. Ongelmana nikotiinireseptori-agonisteja käytettäessä on reseptorissa tapahtuva desensitisaatio. Tällöin reseptori sulkeutuu, eikä aktivoidu vaikka agonistia olisi tarjolla tai sitoutuneena reseptoriin. Varsinkin alfa7-reseptori desensitoituu hyvin nopeasti agonistialtistuksen seurauksena. Reseptorien desensitoituminen voi kliinisessä käytössä aiheuttaa lääkeaineen tehon menetyksen. Perinteisen agonistin sitoutumiskohdan lisäksi nikotiinireseptorissa sijaitsee myös muita sitoutumiskohtia, joita kutsutaan allosteerisiksi sitoutumispaikoiksi. Tutkimuksissa on havaittu, että eräät allosteerisesti sitoutuvat aineet, kuten PNU-120596, voivat vahvistaa agonistin aikaansaamaa vastetta ja/tai estää reseptorin desensitoitumista. Näitä aineita kutsutaan positiivisiksi allosteerisiksi modulaattoreiksi ja niiden ajatellaan olevan vaihtoehto desensitoitumisen aiheuttamaan tehon menetyksen ongelmaan. Nikotiinireseptorien positiivisten allosteeristen modulaattorien tarkkaa vaikutusta ja sitoutumiskohtaa reseptoriin ei vielä tarkkaan tiedetä. Tutkimuksen aiheena oli karakterisoida positiivisten allosteeristen modulaattoreiden vaikutuksia alfa7-nikotiinireseptoriin. Tutkimuksessa tarkoituksena oli käyttää hyväksi laboratoriossa aiemmin tehtyä havaintoa, jonka mukaan alfa7-nikotiinireseptorin transmembraaniosan aminohappoon tehdyn mutaation L247T seurauksena positiiviset allosteeriset modulaattorit muuttuvat agonisteiksi. Haluttiin selvittää, kuinka agonistin sitoutumiskohtaan kohdennettua mutageneesiä käyttäen tehty mutaatio W149M tai W149F vaikuttavat PNU-120596:n kykyyn toimia agonistina alfa7L247T reseptoriin. Asetyylikoliini toimi konventionaalisen agonistin mallina tutkimuksessa. Tutkimuksen toinen tavoite oli tehdä mutaatio M253Lalfa7-reseptorin transmembraaniosaan. Mutaation on todettu estävän allosteeristen potentiaattoreiden kykyä voimistaa agonistin aikaansaamaa vastetta. Tarkoitus oli tutkia millaisia vaikutuksia M253L-mutaatiolla on allosteerisen potentiaattorin kykyyn toimia agonistina L247T-mutaation sisältävään reseptoriin. Mutatoidun reseptorin mRNA mikroinjektoitiin oosyyttiin ja elektrofysiologian avulla tutkittiin ilmennettyjen reseptorien toimintaa käyttäen kahden elektrodin jännitelukitus -menetelmää. Kaikki suunnitellut mutaatiot saatiin tehtyä onnistuneesti alfa7- ja alfa7L247T-reseptoreihin. Ortosteerisen sitoutumiskohdan mutaatio villin tyypin Į7-reseptorissa vaikutti hyvin voimakkaasti joko asetyylikoliinin sitoutumiseen reseptoriin tai reseptorin toimintaan, sillä asetyylikoliinilla ei reseptorista saatu mitattua vasteita. Myöskään PNU-120596 yksinään ei saanut aikaan vasteita alfa7W149M-reseptorissa. Kaksoismutatoidussa alfa7W149M/L247T-reseptorissa puolestaan havaittiin, että asetyylikoliinin annos-vaste -kuvaaja siirtyi huomattavasti enemmän oikealle kuin PNU-120596:n, kun verrattiin annos-vaste –kuvaajia alfa7L247T ja alfa7W149M/L247T–reseptoreiden välillä. Transmembraaniosan mutaatio M253L ei vaikuttanut PNU-120596:n kykyyn toimia agonistina alfa7L247T-reseptoriin, eikä sillä ollut vaikutusta asetyylikoliinin annosvaste-kuvaajiin. Tutkimus tukee aiempia havaintoja siitä, että positiivisten allosteeristen modulaattoreiden sitoutumiskohta nikotiinireseptorissa sijaitsisi transmembraaniosassa. M253L-mutaation osalta tulokset ovat hieman ristiriidassa aiempien tulosten kanssa. L247T-mutaatio vaikuttaa hyvin voimakkaasti nikotiinireseptorin toimintaan sekä sijaitsee aminohapon M253 läheisyydessä. On mahdollista, että se peittää M253L-mutaation vaikutuksen. Toisaalta voi olla, että M253 on aminohappo, joka vaikuttaa vain reseptorivasteiden voimistumiseen eikä allosteeristen potentiaattoreiden sitoutumiseen.