Molecular Determinants of Orexin Receptor Ligand Interaction : Studies on Ligand Selectivity and Impact of Calcium

Neuropeptides orexin-A and orexin-B, and their receptors OX1 and OX2 were first found as regulators of appetite, and later several other functions have been found as well. Orexin peptides and receptors have been researched, especially in the field of drug discovery; however many relevant biochemical properties remain partly unsolved, including the factors determining orexin ligand binding properties and ligand selectivity. The study of these factors was pursued in this thesis. Ligand selectivity was studied with chimaeric orexin receptors. We mapped some of the molecular determinants of orexin receptors that are needed for the selectivity of orexin agonists and OX1-specific antagonist. The second quarter of the orexin receptors seems to be the most important area both for agonist and antagonist selectivity. However, for antagonist selectivity, the third quarter also seems to have a role. Activated orexin receptors cause a strong elevation of intracellular calcium. However, reduction of extracellular calcium attenuates that increase and other orexin receptor signalling, mediated by certain phospholipases and kinases, for instance. It remains unknown, however, how calcium causes these effects. With [125I]-orexin-A, a clear decrease in binding was observed after reduction of the extracellular calcium concentration. Also, we saw a similar reduction in the activities of phospholipase C (PLC) and adenylyl cyclase (AC). The concentration-relationship of calcium was identical for radioligand binding, PLC activation, and AC stimulation, while AC inhibition was more strongly attenuated. When the driving force for calcium influx was reduced with high-K+ medium, the orexin-A-induced PLC activity was more strongly reduced than orexin-A binding. In addition, inhibition of the orexin receptor-operated calcium channels had a more pronounced effect on the PLC activity than on the binding. It is thus suggested that reduction of extracellular calcium concentration both inhibits orexin binding and attenuates enzymatic activity. Orexin-B has higher binding affinity for OX2 than OX1 receptor and [Ala11, D-Leu15]-orexin-B, an orexin-B variant, has been reported to display even higher OX2-selectivity. We observed that [Ala11, D-Leu15]-orexin-B showed much lower OX2-selectivity than originally reported. In addition, the selectivity of both forms of orexin-B was dependent on the cell line. These findings may be caused by biased agonism of the orexin receptor, meaning that the orexin receptor can be found in multiple conformations, each of which can interact differently with an agonist. This result extends our knowledge of orexin ligand binding properties, and the phenomenon should be considered, for instance, when novel agonists for orexin receptors are screened.Neuropeptidien oreksiini-A ja oreksiini-B, ja niiden reseptoreiden OX1 ja OX2, löydettiin aluksi säätelevän ruokahalua, mutta myöhemmin niille on löydetty myös monia muita tehtäviä. Oreksiinipeptidejä ja -reseptoreita tutkitaan etenkin lääkekehityksessä, mutta silti monet tärkeät biokemialliset ominaisuudet, kuten ligandin sitoutumiseen ja selektiivisyyteen vaikuttavat tekijät, ovat osittain selvittämättä. Tässä väitöskirjatyössä tutkittiin näitä tekijöitä. Ligandin selektiivisyyttä tutkittiin kimeerisillä reseptoreilla, joilla kartoitimme oreksiiniagonistien ja OX1-spesifisen antagonistin selektiivisyyteen tarvittavia alueita oreksiinireseptoreissa. Oreksiinireseptoreiden toinen neljännes näyttäisi olevan tärkein alue sekä agonistien että antagonistin selektiivisyydelle. Kuitenkin myös kolmannella neljänneksellä on merkitystä antagonistin selektiivisyydelle. Oreksiinireseptorin aktivoituminen aiheuttaa solunsisäisen kalsiumin lisääntymisen. Tätä, ja mm. tiettyjen fosfolipaasien ja -kinaasien aktiivisuuta, voidaan heikentää vähentämällä solunulkoista kalsiumia. Ei ole kuitenkaan tiedossa, kuinka kalsiumi aiheuttaa nämä vaikutukset. Solunulkoisen kalsiumin vähentäminen vähensi selvästi [125I]-oreksiini-A:n sitoutumista. Vastaavasti, myös fosfolipaasi C:n (PLC) ja adenylaattisyklaasin (AC) aktiivisuudet vähenivät. Kalsiumin vähentäminen heikensi yhtä paljon radioligandin sitoutumista, PLC:n aktivaatiota ja AC:n stimulaatiota, kun taas AC:n inhibitio heikkeni enemmän. Kalsiumin sisäänvirtauksen vähentäminen puskurilla, jossa on paljon K+-ioneja, vähensi enemmän PLC:n aktivoitumista kuin oreksiini-A:n sitoutumista. Lisäksi reseptorin säätelemien kalsium kanavien inhibitiolla oli suurempi vaikutus PLC-aktiivisuuteen kuin sitoutumiseen. Täten vaikuttaisi siltä, että solunulkoisen kalsiumin vähentäminen estää sekä oreksiinin sitoutumista että entsymaattista aktiivisuutta. Oreksiini-B:llä on korkeampi sitoutumisaffiniteetti OX2 kuin OX1 reseptoriin, ja oreksiini-B muunnoksen, [Ala11, D-Leu15]-oreksiini-B:n, on raportoitu olevan vielä enemmän spesifisempi OX2:lle. Huomasimme, että [Ala11, D-Leu15]-oreksiini-B:n OX2-selektiivisyys oli paljon matalampi kuin alun perin on raportoitu. Lisäksi, molempien oreksiini-B muotojen selektiivisyys riippui solulinjasta. Nämä havainnot voivat aiheutua siitä, että oreksiinireseptorilla voi olla useita eri rakenteita, joista kukin vuorovaikuttaa erilailla agonistin kanssa. Tulokset laajentavat tietämystämme oreksiinin sitoutumisominaisuuksista, ja ne pitäisi ottaa huomioon, esim. kun oreksiinireseptorille seulotaan uusia agonisteja

Orexin/hypocretin receptor chimaeras reveal structural features important for orexin peptide distinction

AbstractWe wanted to analyze the basis for the distinction between OX1 and OX2 orexin receptors by the known agonists, orexin-A, orexin-B and Ala11, d-Leu15-orexin-B, of which the latter two show some selectivity for OX2. For this, chimaeric OX1/OX2 and OX2/OX1 orexin receptors were generated. The receptors were transiently expressed in HEK-293 cells, and potencies of the agonists to elicit cytosolic Ca2+ elevation were measured. The results show that the N-terminal regions of the receptor are most important, and the exchange of the area from the C-terminal part of the transmembrane helix 2 to the transmembrane helix 4 is enough to lead to an almost total change of the receptor’s ligand profile

Autocrine Endocannabinoid Signaling through CB 1 Receptors Potentiates OX 1 Orexin Receptor Signaling s

ABSTRACT It has been proposed that OX 1 orexin receptors and CB 1 cannabinoid receptors can form heteromeric complexes, which affect the trafficking of OX 1 receptors and potentiate OX 1 receptor signaling to extracellular signal-regulated kinase (ERK). We have recently shown that OX 1 receptor activity releases high levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG), suggesting an alternative route for OX 1 -CB 1 receptor interaction in signaling, for instance, in retrograde synaptic transmission. In the current study, we set out to investigate this possibility utilizing recombinant Chinese hamster ovary K1 cells. 2-AG released from OX 1 receptor-expressing cells acted as a potent paracrine messenger stimulating ERK activity in neighboring CB 1 receptor-expressing cells. When OX 1 and CB 1 receptors were expressed in the same cells, OX 1 stimulation-induced ERK phosphorylation and activity were strongly potentiated. The potentiation but not the OX 1 response as such was fully abolished by specific inhibition of CB 1 receptors or the enzyme responsible for 2-AG generation, diacylglycerol lipase (DAGL). Although the results do not exclude the previously proposed OX 1 -CB 1 heteromerization, they nevertheless unequivocally identify DAGL-dependent 2-AG generation as the pivotal determinant of the OX 1 -CB 1 synergism and thus suggest a functional rather than a molecular interaction of OX 1 and CB 1 receptors

Autocrine endocannabinoid signaling through CB 1 receptors potentiates OX 1 orexin receptor signaling MOL #80523 2 Running title: CB 1 receptor signaling potentiates OX 1 receptor signaling

Although the results do not exclude the previously proposed OX 1 -CB 1 heteromerization, they nevertheless unequivocally identify diacylglycerol lipase-dependent 2-AG generation as the pivotal determinant of the OX 1 -CB 1 synergism and thus suggest rather a functional than a molecular interaction of OX 1 and CB 1 receptors

Tumor-independent Detection of Inherited Mismatch Repair Deficiency for the Diagnosis of Lynch Syndrome with High Specificity and Sensitivity

Lynch syndrome (LS) is the most common hereditary cancer syndrome. Early diagnosis improves prognosis and reduces health care costs, through existing cancer surveillance methods. The problem is finding and diagnosing the cancer predisposing genetic condition. The current workup involves a complex array of tests that combines family cancer history and clinical phenotypes with tumor characteristics and sequencing data, followed by a challenging task to interpret the found variant(s). On the basis of the knowledge that an inherited mismatch repair (MMR) deficiency is a hallmark of LS, we have developed and validated a functional MMR test, DiagMMR, that detects inherited MMR deficiency directly from healthy tissue without need of tumor and variant information. The validation included 119 skin biopsies collected from clinically pathogenic MMR variant carriers (MSH2, MSH6) and controls, and was followed by a small clinical pilot study. The repair reaction was performed on proteins extracted from primary fibroblasts and the interpretation was based on the MMR capability of the sample in relation to cutoff, which distinguishes MMR proficient (non-LS) from MMR deficient (LS) function. The results were compared with the reference standard (germline NGS). The test was shown to have exceptional specificity (100%) with high sensitivity (89%) and accuracy (97%). The ability to efficiently distinguish LS carriers from controls was further shown with a high area under the receiving operating characteristic (AUROC) value (0.97). This test offers an excellent tool for detecting inherited MMR deficiency linked to MSH2 or MSH6 and can be used alone or with conventional tests to recognize genetically predisposed individuals.Clinical validation of DiagMMR shows high accuracy in distinguishing individuals with hereditary MSH2 or MSH6 MMR deficiency (i.e., LS). The method presented overcomes challenges faced by the complexity of current methods and can be used alone or with conventional tests to improve the ability to recognize genetically predisposed individuals.Peer reviewe