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.

Cholesterol plays a crucial role in cell membrane organization, dynamics and function. Depletion of cholesterol represents a popular approach to explore cholesterol-sensitivity of membrane proteins. An emerging body of literature shows that the consequence of membrane cholesterol depletion often depends on the actual process (acute or chronic), although the molecular mechanism underlying the difference is not clear. Acute depletion, using cyclodextrin-type carriers, is faster relative to chronic depletion, in which inhibitors of cholesterol biosynthesis are used. With the overall goal of addressing molecular differences underlying these processes, we monitored membrane dipole potential under conditions of acute and chronic cholesterol depletion in CHO-K1 cells, using a voltage-sensitive fluorescent dye in dual wavelength ratiometric mode. Our results show that the observed membrane dipole potential exhibits difference under acute and chronic cholesterol depletion conditions, even when cholesterol content was identical. To the best of our knowledge, these results provide, for the first time, molecular insight highlighting differences in dipolar reorganization in these processes. A comprehensive understanding of processes in which membrane cholesterol gets modulated would provide novel insight in its interaction with membrane proteins and receptors, thereby allowing us to understand the role of cholesterol in cellular physiology associated with health and disease.

G protein‐coupled receptors (GPCRs) are cell membrane associated signaling hubs that orchestrate a multitude of cellular functions upon binding to a diverse variety of extracellular ligands. Since GPCRs are integral membrane proteins with seven‐transmembrane domain architecture, their function, organization and dynamics are intimately regulated by membrane lipids, such as cholesterol. Cholesterol is an extensively studied lipids in terms of its effects on GPCR structure and function. One of the possible mechanisms underlying modulation of GPCR function by cholesterol is via specific interaction of GPCRs with membrane cholesterol. These interactions of GPCRs with membrane cholesterol are often attributed to structural features of GPCRs that could facilitate their preferential association with cholesterol. In this backdrop, cholesterol interaction motifs represent putative interaction sites on GPCRs that could facilitate cholesterol‐sensitive function of these receptors. In this review, we provide an overview of cholesterol interaction motifs found in GPCRs, which have been identified through a combination of crystallography, bioinformatics analysis, and functional studies. In addition, we will highlight, using specific examples, why mere presence of a cholesterol interaction motif at a given site may not directly implicate its role in interaction with membrane cholesterol. We therefore believe that experimental approaches, followed by functional analysis of cholesterol sensitivity of GPCRs, would provide a better understanding of the role played by these motifs in cholesterol‐sensitive function. We envision that a comprehensive knowledge of cholesterol interaction sites in GPCRs would allow us to develop a better understanding of GPCR structure‐function paradigm, and could be useful in future therapeutics. This article is categorized under: Models of Systems Properties and Processes > Mechanistic Models Analytical and Computational Methods > Computational Methods Laboratory Methods and Technologies > Macromolecular Interactions, Methods

ATP-binding cassette (ABC) transporters help export various substrates across the cell membrane and significantly contribute to drug resistance. However, a recent study reported an unusual case in which the loss of an ABC transporter in , orf19.4531 (previously named ROA1), increases resistance against antifungal azoles, which was attributed to an altered membrane potential in the mutant strain. To obtain further mechanistic insights into this phenomenon, here we confirmed that the plasma membrane-localized transporter (renamed for consistency with nomenclature) could efflux xenobiotics such as berberine, rhodamine 123, and paraquat. Moreover, a null mutant, NKKY101, displayed increased susceptibility to these xenobiotics. Interestingly, fluorescence recovery after photobleaching (FRAP) results indicated that NKKY101 mutant cells exhibited increased plasma membrane rigidity, resulting in reduced azole accumulation and contributing to azole resistance. Transcriptional profiling revealed that ribosome biogenesis genes were significantly up-regulated in the NKKY101 mutant. As ribosome biogenesis is a well-known downstream phenomenon of target of rapamycin (TOR1) signaling, we suspected a link between ribosome biogenesis and TOR1 signaling in NKKY101. Therefore, we grew NKKY101 cells on rapamycin and observed TOR1 hyperactivation, which leads to Hsp90-dependent calcineurin stabilization and thereby increased azole resistance. This finding was supported by data from a mouse model of systemic infection in which NKKY101 cells led to higher fungal load after fluconazole challenge than wild-type cells. Taken together, our study uncovers a mechanism of azole resistance in , involving increased membrane rigidity and TOR signaling.

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 serotonin1A 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 serotonin1A 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.