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 serotonin1A receptor stably expressed in HEK-293 cells and the cellular machinery involved in receptor internalization and intracellular trafficking. The serotonin1A 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 serotonin1A receptor during endocytosis. Our results, utilizing inhibitors of specific endocytosis pathways and intracellular markers, show that the serotonin1A 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.

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Visceral leishmaniasis is a vector-borne disease caused by an obligate intracellular protozoan parasite Leishmania donovani. The molecular mechanism involved in internalization of Leishmania is poorly understood. The entry of Leishmania involves interaction with the plasma membrane of host cells. We have previously demonstrated the requirement of host membrane cholesterol in the binding and internalization of L. donovani into macrophages. In the present work, we explored the role of the host actin cytoskeleton in leishmanial infection. We observed a dose-dependent reduction in the attachment of Leishmania promastigotes to host macrophages upon destabilization of the actin cytoskeleton by cytochalasin D. This is accompanied by a concomitant reduction in the intracellular amastigote load. We utilized a recently developed high resolution microscopy-based method to quantitate cellular F-actin content upon treatment with cytochalasin D. A striking feature of our results is that binding of Leishmania promastigotes and intracellular amastigote load show close correlation with cellular F-actin level. Importantly, the binding of Escherichia coli remained invariant upon actin destabilization of host cells, thereby implying specific involvement of the actin cytoskeleton in Leishmania infection. To the best of our knowledge, these novel results constitute the first comprehensive demonstration on the specific role of the host actin cytoskeleton in Leishmania infection. Our results could be significant in developing future therapeutic strategies to tackle leishmaniasis.

Endocytosis is a key regulatory mechanism adopted by G protein-coupled receptors (GPCRs) to modulate downstream signaling responses within a stringent spatiotemporal regime. Although the role of membrane lipids has been extensively studied in the context of the function, organization, and dynamics of GPCRs, their role in receptor endocytosis remains largely unexplored. Cholesterol, the predominant sterol in higher eukaryotes, plays a crucial role in maintaining the structure and organization of cell membranes and is involved in essential cellular processes in health and disease. The serotonin 1A receptor is a representative GPCR involved in neuronal development and in neuropsychiatric disorders such as anxiety and depression. We recently combined quantitative flow cytometric and confocal microscopic approaches to demonstrate that the serotonin 1A receptor undergoes clathrin-mediated endocytosis upon agonist stimulation and subsequently traffics along the endosomal recycling pathway. In this work, we show that statin-induced chronic cholesterol depletion switches the endocytic pathway of the serotonin 1A receptor from clathrin-to caveolin-mediated endocytosis. Interestingly, under these conditions, a significant proportion of endocytosed receptors is rerouted toward lysosomal degradation. To the best of our knowledge, these results constitute one of the first comprehensive reports on the role of membrane cholesterol in GPCR endocytosis and trafficking. These results are significant in our overall understanding of the modulatory effects of membrane lipids on GPCR endocytosis and trafficking and could provide novel insight in developing therapeutic interventions against neuropsychiatric disorders such as depression.

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