@article {96415, title = {A molecular sensor for cholesterol in the human serotonin receptor.}, journal = {Sci Adv}, volume = {7}, year = {2021}, month = {2021 Jul}, abstract = {

The function of several G protein-coupled receptors (GPCRs) exhibits cholesterol sensitivity. Cholesterol sensitivity of GPCRs could be attributed to specific sequence and structural features, such as the cholesterol recognition/interaction amino acid consensus (CRAC) motif, that facilitate their cholesterol-receptor interaction. In this work, we explored the molecular basis of cholesterol sensitivity exhibited by the serotonin receptor, the most studied GPCR in the context of cholesterol sensitivity, by generating mutants of key residues in CRAC motifs in transmembrane helix 2 (TM2) and TM5 of the receptor. Our results show that a lysine residue (K101) in one of the CRAC motifs is crucial for sensing altered membrane cholesterol levels. Insights from all-atom molecular dynamics simulations showed that cholesterol-sensitive functional states of the serotonin receptor are associated with reduced conformational dynamics of extracellular loops of the receptor. These results constitute one of the first reports on the molecular mechanism underlying cholesterol sensitivity of GPCRs.

}, issn = {2375-2548}, doi = {10.1126/sciadv.abh2922}, author = {Kumar, G Aditya and Sarkar, Parijat and Stepniewski, Tomasz Maciej and Jafurulla, Md and Singh, Shishu Pal and Selent, Jana and Chattopadhyay, Amitabha} } @article {49144, title = {5-HT deletion protects against Clozapine-induced hyperglycemia.}, journal = {J Pharmacol Sci}, volume = {139}, year = {2019}, month = {2018 Dec 18}, pages = {133-135}, abstract = {

Clozapine is an antipsychotic known for its superior efficacy in treating drug-resistant Schizophrenia. However, Clozapine induces various side effects such as hyperglycemia, agranulocytosis, weight gain etc. The mechanisms of these Clozapine-induced side effects have remained largely elusive though an important role is ascribed to 5-HT (Serotonin receptor subtype-2A). In this pilot study, we report for the first time that the 5-HT {\textquoteright}global{\textquoteright} knockout mice (Htr2a) are resistant to the Clozapine-induced hyperglycemia. Importantly though, the Htr2a mice exhibit near normal basal glucose metabolism in the glucose tolerance tests. Collectively, the Htr2a mice provide an important tool to study the Clozapine-induced hyperglycemia.

}, issn = {1347-8648}, doi = {10.1016/j.jphs.2018.11.015}, author = {Joshi, Radhika Sudhir and Singh, Shishu Pal and Panicker, Mitradas M} } @article {53185, title = {Amniotic membrane as novel scaffold for human iPSC-derived cardiomyogenesis.}, journal = {In Vitro Cell Dev Biol Anim}, year = {2019}, month = {2019 Feb 24}, abstract = {

Recent approaches of using decellularized organ matrices for cardiac tissue engineering prompted us to culture human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) on the human amniotic membrane (hAM). Since hAM has been used lately to patch diseased hearts in patients and has shown anti-inflammatory and anti-fibrotic benefits, it qualifies as a cardiac compatible and clinically relevant heart tissue scaffold. The aim of this study was to test the ability of the hAM to support attachment, differentiation, and maturation of hiPSC-derived CMs in vitro. hAMs were prepared from term placenta. An in-house generated hiPSC line was used for CM derivation. hiPSC-derived cardiac progenitors were cultured on the surface of cryopreserved hAMs and in the presence of cytokines promoting cardiac differentiation. CMs grown on hAM and popular basement membrane matrix (BMM) Matrigel{\texttrademark} were compared for the following aspects of cardiac development: the morphology of cardiomyocytes with respect to shape and cellular alignments, levels of cardiac-related gene transcript expression, functionality in terms of spontaneous calcium fluxes and mitochondrial densities and distributions. hAM is biocompatible with hiPSC-derived CMs. hAM increased cardiac transcription regulator and myofibril protein transcript expression, accelerated intracellular calcium transients, and enhanced cellular mitochondrial complexity of its cardiomyocytes in comparison to cardiomyocytes differentiated on Matrigel{\texttrademark}. Our data suggests that hAM supports differentiation and improves cardiomyogenesis in comparison to Matrigel{\texttrademark}. hAMs are natural, easily and largely available. The method of preparing hAM cardiac sheets described here is simple with potential for clinical transplantation. Graphical abstract A An outline of the differentiation protocol with stage-specific growth factors and culture media used. B Cell fates from pluripotent stem cells to cardiomyocytes during differentiation on the amniotic membrane. C-FPhotomicrographs of cells at various stages of differentiation. Scale bars represent 100\ μm.

}, issn = {1543-706X}, doi = {10.1007/s11626-019-00321-y}, author = {Parveen, Shagufta and Singh, Shishu Pal and Panicker, M M and Gupta, Pawan Kumar} } @article {54875, title = {Exploring Endocytosis and Intracellular Trafficking of the Human Serotonin Receptor.}, journal = {Biochemistry}, year = {2019}, month = {2019 Mar 28}, abstract = {

G protein-coupled receptors (GPCRs) represent the largest class of receptors involved in signal transduction across cell membranes and are major drug targets in all clinical areas. Endocytosis of GPCRs offers a regulatory mechanism for sustaining their signaling within a stringent spatiotemporal regime. In this work, we explored agonist-induced endocytosis of the human serotonin receptor stably expressed in HEK-293 cells and the cellular machinery involved in receptor internalization and intracellular trafficking. The serotonin receptor is a popular GPCR implicated in neuropsychiatric disorders such as anxiety and depression and serves as an important drug target. In spite of its pharmacological relevance, its mechanism of endocytosis and intracellular trafficking is less understood. In this context, we have utilized a combination of robust population-based flow cytometric analysis and confocal microscopic imaging to address the path and fate of the serotonin receptor during endocytosis. Our results, utilizing inhibitors of specific endocytosis pathways and intracellular markers, show that the serotonin receptor undergoes endocytosis predominantly via the clathrin-mediated pathway and subsequently recycles to the plasma membrane via recycling endosomes. These results would enhance our understanding of molecular mechanisms of GPCR endocytosis and could offer novel insight into the underlying mechanism of antidepressants that act via the serotonergic pathway. In addition, our results could be relevant in understanding cell (or tissue)-specific GPCR endocytosis.

}, issn = {1520-4995}, doi = {10.1021/acs.biochem.9b00033}, author = {Kumar, G Aditya and Sarkar, Parijat and Jafurulla, Md and Singh, Shishu Pal and Srinivas, Gunda and Pande, Gopal and Chattopadhyay, Amitabha} }