Predicted signal peptides, and the role of the N-terminal tail, at the monoamine G-protein coupled receptors 5-HT2c and α2c

Background: G-protein coupled receptors (GPCRs) have seven transmembrane helices and are situated in the cell membrane, where they transduce signals from specific ligands to the interior of the cell. The first step in the path toward a functional GPCR is the synthesis and incorporation of the evolving receptor into the endoplasmic reticulum (ER) membrane. This process is named cotranslational translocation and is directed by a hydrophobic signal sequence located either in the N-terminus or in the first transmembrane segment (TM1). When the signal sequence is located in the N-terminus, it is cleaved off after translocation and is called a signal peptide (SP). When the signal sequence is part of the TM1 it is called a signal anchor. Monoamine GPCRs have in general short N-termini and are expected to use their TM1 as a signal anchor. Two monoamine GPCRs are nevertheless predicted by a SP prediction software to have signal peptides: The 5-HT2C receptor and the α2C-adrenoceptor. For the 5-HT2C receptor the consequence of having the predicted SP is that a single nucleotide polymorphism (SNP) will not be present in the mature receptor in the cell membrane. This SNP (Cys23Ser) has in several studies been associated with numerous clinical conditions and outcomes of pharmacotherapy. The α2C-adrenoceptor is poorly expressed at the cell surface and has a large intracellular pool of receptors. It has been shown previously for other receptors that by adding a cleavable signal peptide sequence immediately upstream to the endogenous Nterminus, the expression levels of β2- and α1D-adrenoceptor are greatly enhanced. Consequently, it is seemingly odd that the poorly expressed α2C-adrenoceptor is predicted to contain a SP. Objective: The primary aim was to determine whether the monoamine GPCRs 5- HT2C and α2C have cleavable signal peptides as predicted. A secondary aim was to determine what relevance the N-termini of the 5-HT2C and α2C receptors have for expression levels of the receptors. Materials and methods: Methods included engineering receptor constructs and chimeras by PCR and transiently transfecting COS-7 and HEK293 cells. Receptor constructs containing FLAG epitope were investigated with the primary antibodies M1 and M2 in epifluorescence and confocal microscopy. Expression levels of wild type and rebuilt receptor constructs were determined by radioligand binding performed on membrane preparations. For α2C-adrenoceptors radioligand binding was also performed on whole cells, matching the membranes, to exclude binding to an intracellular pool of receptors. Results and Conclusions: The 5-HT2C receptor has a 32 amino acid long cleavable signal peptide, as predicted by its amino acid sequence. When the signal peptide is made non-cleavable by changing one amino acid, the expression level of the receptor is reduced by 70%. We therefore conclude that a 32 amino acid long cleavable signal peptide is participating in the integration of the 5-HT2C receptor into the ER membrane. Consequently, the mature receptor does not contain the aforementioned Cys23Ser SNP. The α2C-adrenoceptor does not possess a 22 amino acid long cleavable signal peptide. Among the α2C-adrenoceptor constructs, expression was highest for the wild type receptor where the endogenous N-terminus was retained. Furthermore, all attempts at increasing the expression level of the α2C-adrenoceptor by adding a known SP or by truncating the N-tail, failed. We conclude that the Nterminus is not a major contributor to the low expression level of the α2C-adrenoceptor

Quetiapine, Misuse and dependency: A case-series of questions to a Norwegian network of drug information centers

Purpose: The second-generation antipsychotic quetiapine has been associated with misuse and dependency. We aimed to review questions to the Norwegian network of drug information centers concerning this potential drug safety problem. Methods: We conducted a Boolean search in the database of the Regional Medicines Information and Pharmacovigilance Centres in Norway (RELIS) combining the indexed categories “quetiapine” and “adverse drug reaction” with the text words “misuse” or “dependency”. Question–answer pairs (Q/As) in the full-text, searchable RELIS database were defined as cases. Cases were analyzed for drug safety issues linked to use of quetiapine, including off-label use, polypharmacy and other patient risk factors. Results: The search resulted in 54 cases. Forty-six cases (85%) were patient-related, and a majority came from physicians working in hospitals. Twenty-nine cases (54%) concerned patients with a history of addiction, 14 cases (26%) had polypharmacy, and off-label use of quetiapine for insomnia was identified in 14 of the cases (26%). Only three of the cases included a specific question about patient dependency of quetiapine, and these cases were all associated with insomnia. Conclusion: We conclude that our case series from the Norwegian network of drug information centres reflects that quetiapine frequently involves clinical narratives of a history of addiction, polypharmacy or insomnia (off-label use). However, the case series did not reveal new information about the drug’s addictive potential.publishedVersio

Quetiapine, Misuse and dependency: A case-series of questions to a Norwegian network of drug information centers

Purpose: The second-generation antipsychotic quetiapine has been associated with misuse and dependency. We aimed to review questions to the Norwegian network of drug information centers concerning this potential drug safety problem. Methods: We conducted a Boolean search in the database of the Regional Medicines Information and Pharmacovigilance Centres in Norway (RELIS) combining the indexed categories “quetiapine” and “adverse drug reaction” with the text words “misuse” or “dependency”. Question–answer pairs (Q/As) in the full-text, searchable RELIS database were defined as cases. Cases were analyzed for drug safety issues linked to use of quetiapine, including off-label use, polypharmacy and other patient risk factors. Results: The search resulted in 54 cases. Forty-six cases (85%) were patient-related, and a majority came from physicians working in hospitals. Twenty-nine cases (54%) concerned patients with a history of addiction, 14 cases (26%) had polypharmacy, and off-label use of quetiapine for insomnia was identified in 14 of the cases (26%). Only three of the cases included a specific question about patient dependency of quetiapine, and these cases were all associated with insomnia. Conclusion: We conclude that our case series from the Norwegian network of drug information centres reflects that quetiapine frequently involves clinical narratives of a history of addiction, polypharmacy or insomnia (off-label use). However, the case series did not reveal new information about the drug’s addictive potential

Analysis of questions about use of drugs in breastfeeding to Norwegian drug information centres

Abstract Background Health professionals may advise women to either stop breastfeeding or drug treatment due to restrictive advice in drug monographs. Regional medicines information and pharmacovigilance centres in Norway (RELIS) provide free and industry-independent answers to questions about drugs and breastfeeding documented in a full-text, searchable database (RELIS database). We used the RELIS database to describe which health care practitioners sought information about medication safety in lactation, most common drugs involved, advice provided and which resources were used to provide the advice. Methods A random selection of 100 question-answer pairs (QAPs) from the RELIS database indexed with “BREASTFEEDING” in the period from January 2011 to December 2015 was analysed. Inclusion criteria were queries from health professionals about drugs. Questions about herbal supplements and other exposures not classified as drugs were excluded. The QAPs were manually analysed for compatibility of one or several drugs with breastfeeding, health care profession and workplace of enquirer in addition to advice and search strategy used. Results In the 100 QAPs there were enquires about 152 drugs. Seventy-four questions concerned a single drug, but the number of drugs evaluated varied between 1 and 16. Fifty-nine questions were from physicians, 34 from nurses or midwives, two from pharmacists and two from other health professionals. Questions from physicians contained 93 drug evaluations (61%), nurses or midwives 47 (31%) and pharmacists seven (5%). The most frequent categories of drugs were antidepressants, antiepileptics and immunosuppressants. The most asked about drugs were lamotrigine, codeine, quetiapine and escitalopram. Fifty-nine percent of the drugs were deemed safe while breastfeeding, 16% if precautions were taken and 12% not recommended. Thirty-nine percent of the drug evaluations used an advanced literature search strategy, and this was significantly (p < 0.05) more likely when the enquirer was a physician. Conclusions This analysis of questions to Norwegian medicines information centres about medicine use in breastfeeding indicates the need for communication about safety of drugs affecting the nervous system, primarily to medical doctors and midwives. In the majority of cases the medicine information centre can reassure about the safety of breastfeeding while taking a drug

A new, simple and robust radioligand binding method used to determine kinetic off-rate constants for unlabeled ligands. Application at alpha(2A)- and alpha(2C)-adrenoceptors

Kinetic on and off rate constants for many receptor ligands are difficult to determine with regular radioligand binding technique since only few of the ligands are available in labeled form. Here we developed a new and simple radioligand binding method for determining the kinetic off-rate constant for unlabeled ligands, using whole cells expressing alpha(2A)- and alpha(2C)-adrenoceptors. The new method involves pre-incubation with unlabeled ligand, centrifugation of microtiter plates in order to adhere the cells to the bottom surface, and then upside-down centrifugation of the plates for few seconds to wash away the non-bound fraction of the pre-incubated ligand. The final on-reaction assay for the radioligand is then started by quick addition of a relatively fast-associating radioligand to the cells. The curve obtained is defined by a fairly simple mathematical formula that reflects the simultaneous dissociation of pre-incubated ligand and association of the radioligand. The method proved to produce highly reproducible results in determining the k(off) constants for various unlabeled ligands. The results show that the alpha(2C)-selectivity of MK912 depends mainly on a very slow off-rate at the alpha(2C)-adrenoceptor subtype. Regarding the markedly alpha(2C)- over alpha(2A)-selective compound spiroxatrine, its much faster on-rate at alpha(2C)- than alpha(2A)-adrenoceptors explains much of its exceptional alpha(2C)-selectivity. Several new techniques for determining the kinetic component of ligand-receptor interactions at molecular level are currently developing. As a reference, based on standard radioligand binding techniques, the present study describes a simple and robust experimental and mathematical procedure for determining k(off) constants of unlabeled drugs