G Protein-coupled Receptors Endocytosis Research Articles

DANGER Signals Activate G -Protein Receptor Kinases Suppressing Neutrophil Function and Predisposing to Infection After Tissue Trauma.

Injured tissue predisposes the subject to local and systemic infection. We studied injury-induced immune dysfunction seeking novel means to reverse such predisposition. Injury mobilizes primitive "DANGER signals" [danger-associated molecular patterns (DAMPs)] activating innate immunocyte (neutrophils, PMN) signaling and function. Mitochondrial formyl peptides activate G -protein coupled receptors (GPCR) like formyl peptide receptor-1. Mitochondrial DNA and heme activate toll-like receptors (TLR9 and TLR2/4). GPCR kinases (GRKs) can regulate GPCR activation. We studied human and mouse PMN signaling elicited by mitochondrial DAMPs (GPCR surface expression; protein phosphorylation, or acetylation; Ca 2+ flux) and antimicrobial functions [cytoskeletal reorganization, chemotaxis (CTX), phagocytosis, bacterial killing] in cellular systems and clinical injury samples. Predicted rescue therapies were assessed in cell systems and mouse injury-dependent pneumonia models. Mitochondrial formyl peptides activate GRK2, internalizing GPCRs and suppressing CTX. Mitochondrial DNA suppresses CTX, phagocytosis, and killing through TLR9 through a novel noncanonical mechanism that lacks GPCR endocytosis. Heme also activates GRK2. GRK2 inhibitors like paroxetine restore functions. GRK2 activation through TLR9 prevented actin reorganization, implicating histone deacetylases (HDACs). Actin polymerization, CTX, bacterial phagocytosis, and killing were also rescued, therefore, by the HDAC inhibitor valproate. Trauma repository PMN showed GRK2 activation and cortactin deacetylation, which varied with severity and was most marked in patients developing infections. Either GRK2 or HDAC inhibition prevented loss of mouse lung bacterial clearance, but only the combination rescued clearance when given postinjury. Tissue injury-derived DAMPs suppress antimicrobial immunity through canonical GRK2 activation and a novel TLR-activated GRK2-pathway impairing cytoskeletal organization. Simultaneous GRK2/HDAC inhibition rescues susceptibility to infection after tissue injury.

Metabolic depletion of sphingolipids inhibits agonist-induced endocytosis of the serotonin1A receptor.

G protein-coupled receptors (GPCRs) are vital cellular signaling machinery and currently represent ~40% drug targets. Endocytosis of GPCRs is an important process that allows stringent spatiotemporal control over receptor population on the cell surface. Although the role of proteins in GPCR endocytosis is well addressed, the contribution of membrane lipids in this process is rather unexplored. Sphingolipids are essential functional lipids in higher eukaryotes and are implicated in several neurological functions. To understand the role of sphingolipids in GPCR endocytosis, we subjected cells expressing human serotonin1A receptors (an important neurotransmitter GPCR involved in cognitive and behavioral functions) to metabolic sphingolipid depletion using fumonisin B1 , an inhibitor of sphingolipid biosynthetic pathway. Our results, using flow cytometric analysis and confocal microscopic imaging, show that sphingolipid depletion inhibits agonist-induced endocytosis of the serotonin1A receptor in a concentration-dependent manner, which was restored when sphingolipid levels were replenished. We further show that there was no change in the internalization of transferrin, a marker for clathrin-mediated endocytosis, under sphingolipid-depleted condition, highlighting the specific requirement of sphingolipids for endocytosis of serotonin1A receptors. Our results reveal the regulatory role of sphingolipids in GPCR endocytosis and highlight the importance of neurotransmitter receptor trafficking in health and disease.

Surveying nonvisual arrestins reveals allosteric interactions between functional sites.

Arrestins are important scaffolding proteins that are expressed in all vertebrate animals. They regulate cell-signaling events upon binding to active G-protein coupled receptors (GPCR) and trigger endocytosis of active GPCRs. While many of the functional sites on arrestins have been characterized, the question of how these sites interact is unanswered. We used anisotropic network modeling (ANM) together with our covariance compliment techniques to survey all the available structures of the nonvisual arrestins to map how structural changes and protein-binding affect their structural dynamics. We found that activation and clathrin binding have a marked effect on arrestin dynamics, and that these dynamics changes are localized to a small number of distant functional sites. These sites include α-helix 1, the lariat loop, nuclear localization domain, and the C-domain β-sheets on the C-loop side. Our techniques suggest that clathrin binding and/or GPCR activation of arrestin perturb the dynamics of these sites independent of structural changes.

GPCR/endocytosis/ERK signaling/S2R is involved in the regulation of the internalization, mitochondria-targeting and -activating properties of human salivary histatin 1

Human salivary histatin 1 (Hst1) exhibits a series of cell-activating properties, such as promoting cell spreading, migration, and metabolic activity. We recently have shown that fluorescently labeled Hst1 (F-Hst1) targets and activates mitochondria, presenting an important molecular mechanism. However, its regulating signaling pathways remain to be elucidated. We investigated the influence of specific inhibitors of G protein-coupled receptors (GPCR), endocytosis pathways, extracellular signal-regulated kinases 1/2 (ERK1/2) signaling, p38 signaling, mitochondrial respiration and Na+/K+-ATPase activity on the uptake, mitochondria-targeting and -activating properties of F-Hst1. We performed a siRNA knockdown (KD) to assess the effect of Sigma-2 receptor (S2R) /Transmembrane Protein 97 (TMEM97)—a recently identified target protein of Hst1. We also adopted live cell imaging to monitor the whole intracellular trafficking process of F-Hst1. Our results showed that the inhibition of cellular respiration hindered the internalization of F-Hst1. The inhibitors of GPCR, ERK1/2, phagocytosis, and clathrin-mediated endocytosis (CME) as well as siRNA KD of S2R/TMEM97 significantly reduced the uptake, which was accompanied by the nullification of the promoting effect of F-Hst1 on cell metabolic activity. Only the inhibitor of CME and KD of S2R/TMEM97 significantly compromised the mitochondria-targeting of Hst1. We further showed the intracellular trafficking and targeting process of F-Hst1, in which early endosome plays an important role. Overall, phagocytosis, CME, GPCR, ERK signaling, and S2R/TMEM97 are involved in the internalization of Hst1, while only CME and S2R/TMEM97 are critical for its subcellular targeting. The inhibition of either internalization or mitochondria-targeting of Hst1 could significantly compromise its mitochondria-activating property.

Cholesterol-dependent endocytosis of GPCRs: implications in pathophysiology and therapeutics.

G protein-coupled receptors (GPCRs) are the largest family of transmembrane proteins that relay extracellular signals across the plasma membrane and elicit an intricate cascade of cellular signaling events. A significantly large fraction of available drugs target GPCRs in order to exert fine control over functional outcomes from these receptors in pathological conditions. In this context, endocytosis and intracellular trafficking of GPCRs stringently regulate signaling outcomes from GPCRs within physiologically relevant spatiotemporal regimes. The membrane microenvironment around GPCRs has recently emerged as a key player in receptor function. Cholesterol is the single most abundant lipid in the eukaryotic plasma membrane and plays a central role in membrane organization and dynamics, with far-reaching functional implications in cellular physiology. In this review, we discuss current excitements in GPCR endocytosis and trafficking, with an emphasis on the role of membrane cholesterol. We envision that a detailed understanding of the contribution of membrane lipids such as cholesterol in spatiotemporal regulation of GPCR signaling would enable the development of therapeutic interventions fine-tuned to receptors residing in specific membrane microenvironments.

Discovery of a dual Ras and ARF6 inhibitor from a GPCR endocytosis screen

Internalization and intracellular trafficking of G protein-coupled receptors (GPCRs) play pivotal roles in cell responsiveness. Dysregulation in receptor trafficking can lead to aberrant signaling and cell behavior. Here, using an endosomal BRET-based assay in a high-throughput screen with the prototypical GPCR angiotensin II type 1 receptor (AT1R), we sought to identify receptor trafficking inhibitors from a library of ~115,000 small molecules. We identified a novel dual Ras and ARF6 inhibitor, which we named Rasarfin, that blocks agonist-mediated internalization of AT1R and other GPCRs. Rasarfin also potently inhibits agonist-induced ERK1/2 signaling by GPCRs, and MAPK and Akt signaling by EGFR, as well as prevents cancer cell proliferation. In silico modeling and in vitro studies reveal a unique binding modality of Rasarfin within the SOS-binding domain of Ras. Our findings unveil a class of dual small G protein inhibitors for receptor trafficking and signaling, useful for the inhibition of oncogenic cellular responses.

β-arrestin–dependent PI(4,5)P2 synthesis boosts GPCR endocytosis

β-arrestins regulate many cellular functions including intracellular signaling and desensitization of G protein-coupled receptors (GPCRs). Previous studies show that β-arrestin signaling and receptor endocytosis are modulated by the plasma membrane phosphoinositide lipid phosphatidylinositol-(4, 5)-bisphosphate (PI(4,5)P2). We found that β-arrestin also helped promote synthesis of PI(4,5)P2 and up-regulated GPCR endocytosis. We studied these questions with the Gq-coupled protease-activated receptor 2 (PAR2), which activates phospholipase C, desensitizes quickly, and undergoes extensive endocytosis. Phosphoinositides were monitored and controlled in live cells using lipid-specific fluorescent probes and genetic tools. Applying PAR2 agonist initiated depletion of PI(4,5)P2, which then recovered during rapid receptor desensitization, giving way to endocytosis. This endocytosis could be reduced by various manipulations that depleted phosphoinositides again right after phosphoinositide recovery: PI(4)P, a precusor of PI(4,5)P2, could be depleted at either the Golgi or the plasma membrane (PM) using a recruitable lipid 4-phosphatase enzyme and PI(4,5)P2 could be depleted at the PM using a recruitable 5-phosphatase. Endocytosis required the phosphoinositides. Knock-down of β-arrestin revealed that endogenous β-arrestin normally doubles the rate of PIP5-kinase (PIP5K) after PAR2 desensitization, boosting PI(4,5)P2-dependent formation of clathrin-coated pits (CCPs) at the PM. Desensitized PAR2 receptors were swiftly immobilized when they encountered CCPs, showing a dwell time of ∼90 s, 100 times longer than for unactivated receptors. PAR2/β-arrestin complexes eventually accumulated around the edges or across the surface of CCPs promoting transient binding of PIP5K-Iγ. Taken together, β-arrestins can coordinate potentiation of PIP5K activity at CCPs to induce local PI(4,5)P2 generation that promotes recruitment of PI(4,5)P2-dependent endocytic machinery.

Arrestin-Dependent and -Independent Internalization of G Protein-Coupled Receptors: Methods, Mechanisms, and Implications on Cell Signaling.

Agonist-induced endocytosis is a key regulatory mechanism for controlling the responsiveness of the cell by changing the density of cell surface receptors. In addition to the role of endocytosis in signal termination, endocytosed G protein-coupled receptors (GPCRs) have been found to signal from intracellular compartments of the cell. Arrestins are generally believed to be the master regulators of GPCR endocytosis by binding to both phosphorylated receptors and adaptor protein 2 (AP-2) or clathrin, thus recruiting receptors to clathrin-coated pits to facilitate the internalization process. However, many other functions have been described for arrestins that do not relate to their role in terminating signaling. Additionally, there are now more than 30 examples of GPCRs that internalize independently of arrestins. Here we review the methods, pharmacological tools, and cellular backgrounds used to determine the role of arrestins in receptor internalization, highlighting their advantages and caveats. We also summarize key examples of arrestin-independent GPCR endocytosis in the literature and their suggested alternative endocytosis pathway (e.g., the caveolae-dependent and fast endophilin-mediated endocytosis pathways). Finally, we consider the possible function of arrestins recruited to GPCRs that are endocytosed independently of arrestins, including the catalytic arrestin activation paradigm. Technological improvements in recent years have advanced the field further, and, combined with the important implications of endocytosis on drug responses, this makes endocytosis an obvious parameter to include in molecular pharmacological characterization of ligand-GPCR interactions. SIGNIFICANCE STATEMENT: G protein-coupled receptor (GPCR) endocytosis is an important means to terminate receptor signaling, and arrestins play a central role in the widely accepted classical paradigm of GPCR endocytosis. In contrast to the canonical arrestin-mediated internalization, an increasing number of GPCRs are found to be endocytosed via alternate pathways, and the process appears more diverse than the previously defined "one pathway fits all."

Membrane cholesterol regulates endocytosis and trafficking of the serotonin1A receptor: Insights from acute cholesterol depletion

Endocytosis and intracellular trafficking constitute important regulatory features associated with G protein-coupled receptor (GPCR) function. GPCR endocytosis involves several remodeling events at the plasma membrane orchestrated by a concerted interplay of a large number of proteins and membrane lipids. Although considerable literature exists on the protein framework underlying GPCR endocytosis, the role of membrane lipids in this process remains largely unexplored. In order to explore the role of membrane cholesterol (an essential and important lipid in higher eukaryotes) in GPCR endocytosis, we monitored the effect of acute cholesterol depletion using methyl-β-cyclodextrin (MβCD) on endocytosis and intracellular trafficking of the serotonin1A receptor, an important neurotransmitter GPCR. Our results show that the serotonin1A receptor exhibits agonist-induced clathrin-mediated endocytosis with a concentration-dependent inhibition in internalization with increasing concentrations of MβCD, which was restored upon cholesterol replenishment. Interestingly, subsequent to internalization under these conditions, serotonin1A receptors were re-routed toward lysosomal degradation, instead of endosomal recycling observed under normal conditions, thereby implicating membrane cholesterol in modulation of intracellular trafficking of the receptor. This raises the possibility of a novel cholesterol-dependent role of intracellular sorting proteins in GPCR trafficking. These results differ from our previous observations on the endocytosis of the serotonin1A receptor upon statin-induced chronic cholesterol depletion, in terms of endocytic pathway. We conclude that analysis of complex cellular trafficking events such as GPCR endocytosis under acute and chronic cholesterol depletion conditions should be carried out with caution due to fundamental differences underlying these processes.

Protein Phosphatase 2A and Clathrin-Mediated Endocytosis Facilitate Robust Melanopsin Light Responses and Resensitization.

PurposeIntrinsically photosensitive retinal ganglion cells (ipRGCs) that express the visual pigment melanopsin regulate non-image-forming visual tasks, such as circadian photoentrainment and pupil constriction, as well as contrast detection for image formation. Sustained ipRGC function throughout the day is, therefore, of great importance. Melanopsin is a bistable rhabdomeric-type (R-type) visual pigment, which is thought to use light to regenerate its chromophore from all-trans-retinal back to 11-cis-retinal and does not depend on constant chromophore supply to the extent required by visual pigment in rod and cone photoreceptors. Like the majority of photopigments and G-protein-coupled receptors (GPCRs), melanopsin deactivation requires C-terminal phosphorylation and subsequent β-arrestin binding. We hypothesize that melanopsin utilizes canonical GPCR resensitization mechanisms, including dephosphorylation and endocytosis, during the light, and together, they provide a mechanism for prolonged light responses.MethodsHere, we examined expression of protein phosphatases from a variety of subfamilies by RT-PCR and immunohistochemical analyses of the mouse retina. The expression of protein phosphatase 2A (PP2A) in ipRGCs was assessed. We also examine the role of phosphatase and endocytic activity in sustaining melanopsin signaling using transiently-transfected HEK293 cells.ResultsOur analyses suggest that melanopsin-mediated light responses can be rapidly and extensively enhanced by PP2A activity. Light-activated melanopsin undergoes endocytosis in a clathrin-dependent manner. This endocytic activity enhances light responses upon repeated stimulation, implicating a role for endocytic activity in resensitization.ConclusionsThus, we propose that melanopsin phototransduction is maintained by utilizing canonical GPCR resensitization mechanisms rather than reliance on chromophore replenishment from supporting cells.

GRK2-mediated receptor phosphorylation and Mdm2-mediated β-arrestin2 ubiquitination drive clathrin-mediated endocytosis of G protein-coupled receptors

Clathrin-mediated and caveolar endocytic pathways represent the major routes through which G protein-coupled receptors (GPCRs) could be internalized. GPCR kinase 2 (GRK2) and β-arrestins are representative proteins that mediate the GPCR endocytosis. However, the molecular mechanisms through which GRK2 and β-arrestin mediate clathrin-mediated and caveolar endocytosis remain unclear. In this study, we determined the cellular components and processes that mediate the selective interaction between clathrin/caveolin1 and GRK2/β-arrestins. For this we utilized the following: (i) mutant dopamine D2 receptor and β2 adrenoceptor in which the potential GRK2 phosphorylation sites were altered and (ii) cells in which clathrin, caveolin1, β-arrestins, or Mdm2 expression were knocked down. Our results showed that clathrin-mediated endocytosis occurs more rapidly than caveolar endocytosis. Clathrin-mediated endocytosis and the interaction between clathrin and GRK2/β-arrestin2 occurred in a GRK2-mediated receptor phosphorylation-dependent manner. In contrast, caveolar endocytosis and the interaction between caveolin1 and GRK2/β-arrestin2 were independent of receptor phosphorylation status. Mdm2-mediated ubiquitination of β-arrestin, which occurred in a receptor phosphorylation-dependent manner, was required for the interaction of arrestin with clathrin. Thus, this study shows that GRK2-mediated receptor phosphorylation accompanied by β-arrestin ubiquitination is a critical cellular event that links GRK2 and β-arrestins to clathrin-mediated endocytosis.

Barbadin selectively modulates FPR2-mediated neutrophil functions independent of receptor endocytosis

FPR2, a member of the family of G protein-coupled receptors (GPCRs), mediates neutrophil migration, a response that has been linked to β-arrestin recruitment. β-Arrestin regulates GPCR endocytosis and can also elicit non-canonical receptor signaling. To determine the poorly understood role of β-arrestin in FPR2 endocytosis and in NADPH-oxidase activation in neutrophils, Barbadin was used as a research tool in this study. Barbadin has been shown to bind the clathrin adaptor protein (AP2) and thereby prevent β-arrestin/AP2 interaction and β-arrestin-mediated GPCR endocytosis. In agreement with this, AP2/β-arrestin interaction induced by an FPR2-specific agonist was inhibited by Barbadin. Unexpectedly, however, Barbadin did not inhibit FPR2 endocytosis, indicating that a mechanism independent of β-arrestin/AP2 interaction may sustain FPR2 endocytosis. This was confirmed by the fact, that FPR2 also underwent agonist-promoted endocytosis in β-arrestin deficient cells, albeit at a diminished level as compared to wild type cells. Dissection of the Barbadin effects on FPR2-mediated neutrophil functions including NADPH-oxidase activation mediated release of reactive oxygen species (ROS) and chemotaxis revealed that Barbadin had no effect on chemotactic migration whereas the release of ROS was potentiated/primed. The effect of Barbadin on ROS production was reversible, independent of β-arrestin recruitment, and similar to that induced by latrunculin A. Taken together, our data demonstrate that endocytic uptake of FPR2 occurs independently of β-arrestin, while Barbadin selectively augments FPR2-mediated ROS production independently of receptor endocytosis. Given that Barbadin binds to AP2 and prevents the AP2/β-arrestin interaction, our results indicate a role for AP2 in FPR2-mediated ROS release from neutrophils.

Genetically encoded intrabody sensors report the interaction and trafficking of β-arrestin 1 upon activation of G-protein–coupled receptors

Agonist stimulation of G-protein-coupled receptors (GPCRs) typically leads to phosphorylation of GPCRs and binding to multifunctional proteins called β-arrestins (βarrs). The GPCR-βarr interaction critically contributes to GPCR desensitization, endocytosis, and downstream signaling, and GPCR-βarr complex formation can be used as a generic readout of GPCR and βarr activation. Although several methods are currently available to monitor GPCR-βarr interactions, additional sensors to visualize them may expand the toolbox and complement existing methods. We have previously described antibody fragments (FABs) that recognize activated βarr1 upon its interaction with the vasopressin V2 receptor C-terminal phosphopeptide (V2Rpp). Here, we demonstrate that these FABs efficiently report the formation of a GPCR-βarr1 complex for a broad set of chimeric GPCRs harboring the V2R C terminus. We adapted these FABs to an intrabody format by converting them to single-chain variable fragments and used them to monitor the localization and trafficking of βarr1 in live cells. We observed that upon agonist simulation of cells expressing chimeric GPCRs, these intrabodies first translocate to the cell surface, followed by trafficking into intracellular vesicles. The translocation pattern of intrabodies mirrored that of βarr1, and the intrabodies co-localized with βarr1 at the cell surface and in intracellular vesicles. Interestingly, we discovered that intrabody sensors can also report βarr1 recruitment and trafficking for several unmodified GPCRs. Our characterization of intrabody sensors for βarr1 recruitment and trafficking expands currently available approaches to visualize GPCR-βarr1 binding, which may help decipher additional aspects of GPCR signaling and regulation.

Statin-Induced Chronic Cholesterol Depletion Switches GPCR Endocytosis and Trafficking: Insights from the Serotonin1A Receptor.

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 serotonin1A 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 serotonin1A 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 serotonin1A 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.

Clathrin and GRK2/3 inhibitors block δ-opioid receptor internalization in myenteric neurons and inhibit neuromuscular transmission in the mouse colon

Endocytosis is a major mechanism through which cellular signaling by G protein-coupled receptors (GPCRs) is terminated. However, recent studies demonstrate that GPCRs are internalized in an active state and continue to signal from within endosomes, resulting in effects on cellular function that are distinct to those arising at the cell surface. Endocytosis inhibitors are commonly used to define the importance of GPCR internalization for physiological and pathophysiological processes. Here, we provide the first detailed examination of the effects of these inhibitors on neurogenic contractions of gastrointestinal smooth muscle, a key preliminary step to evaluate the importance of GPCR endocytosis for gut function. Inhibitors of clathrin-mediated endocytosis (Pitstop2, PS2) or G protein-coupled receptor kinase-2/3-dependent phosphorylation (Takeda compound 101, Cmpd101), significantly reduced GPCR internalization. However, they also attenuated cholinergic contractions through different mechanisms. PS2 abolished contractile responses by colonic muscle to SNC80 and morphine, which strongly and weakly internalize δ-opioid and μ-opioid receptors, respectively. PS2 did not affect the increased myogenic contractile activity following removal of an inhibitory neural influence (tetrodotoxin) but suppressed electrically evoked neurogenic contractions. Ca2+ signaling by myenteric neurons in response to exogenous ATP was unaffected by PS2, suggesting inhibitory actions on neurotransmitter release rather than neurotransmission. In contrast, Cmpd101 attenuated contractions to the cholinergic agonist carbachol, indicating direct effects on smooth muscle. We conclude that, although PS2 and Cmpd101 are effective blockers of GPCR endocytosis in enteric neurons, these inhibitors are unsuitable for the study of neurally mediated gut function due to their inhibitory effects on neuromuscular transmission and smooth muscle contractility.NEW & NOTEWORTHY Internalization of activated G protein-coupled receptors is a major determinant of the type and duration of subsequent downstream signaling events. Inhibitors of endocytosis effectively block opioid receptor internalization in enteric neurons. The clathrin-dependent endocytosis inhibitor Pitstop2 blocks effects of opioids on neurogenic contractions of the colon in an internalization-independent manner. These inhibitors also significantly impact cholinergic neuromuscular transmission. We conclude that these tools are unsuitable for examination of the contribution of neuronal G protein-coupled receptor endocytosis to gastrointestinal motility.

Exploring Endocytosis and Intracellular Trafficking of the Human Serotonin1A Receptor.

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.

Super-Resolution Imaging of G Protein-Coupled Receptors Using Ground State Depletion Microscopy.

G protein-coupled receptors (GPCRs) comprise the largest family of integral membrane proteins, which are coupled to heterotrimeric G proteins to influence cell signaling. Subsequent to G protein activation, agonist-stimulated G protein-coupled receptor kinase (GRK) phosphorylation results in the recruitment of β-arrestin proteins, which form both stable and unstable complexes with GPCRs. β-Arrestins when bound to GPCRs not only contribute to the uncoupling of G protein signaling but also tothe redistribution of GPCRs to clathrin-coated pits via their association with both clathrin and β2-adaptin facilitating GPCR endocytosis. This allows β-arrestins to couple GPCRs to additional cell signaling proteins allowing a second wave of receptor signaling. Importantly, the β-arrestin-regulated subcellular localization of these complexes also plays a critical role in regulating how these signals are transduced and which proteins are recruited. Here, we describe a methodology for assessing the GPCR subcellular localization by super-resolution microscopy and suggest that this methodology can be extended to the study of GPCR/protein complexes.

Measuring surface expression and endocytosis of GPCRs using whole-cell ELISA.

G protein-coupled receptors (GPCRs) constitute a large class of cell surface receptors that recognize a wide array of ligands and mediate a diverse spectrum of signaling pathways. Measuring their surface expression in cellular context is a critical aspect of studying their signaling pathways and cellular outcomes. Upon addition of agonist, GPCRs typically undergo internalization and traffic from the plasma membrane to endosomal compartments. Although radioligand binding has been the primary assay to measure GPCR surface expression and internalization, whole-cell ELISA has now emerged as a powerful alternative approach. Here, we present a step-by-step whole-cell ELISA protocol for measuring relative surface expression and agonist-induced internalization of GPCRs containing engineered N-terminal epitope tag and recombinantly expressed in heterologous cells.

A synthetic intrabody-based selective and generic inhibitor of GPCR endocytosis.

β-arrestins (βarrs) critically mediate desensitization, endocytosis and signaling of G Protein-Coupled Receptors (GPCRs), and they scaffold a large number of interaction partners. However, allosteric modulation of their scaffolding abilities and direct targeting of their interaction interfaces to selectively modulate GPCR functions have not been fully explored yet. Here, we have identified a series of synthetic antibody fragments (Fabs) against different conformations of βarrs from phage display libraries. Several of these Fabs allosterically and selectively modulated the interaction of βarrs with clathrin and ERK MAP kinase. Interestingly, one of these Fabs selectively disrupted βarr-clathrin interaction, and when expressed as an intrabody, it robustly inhibited agonist-induced endocytosis of a broad set of GPCRs without affecting ERK MAP kinase activation. Our data therefore demonstrate the feasibility of selectively targeting βarr interactions using intrabodies and provide a novel framework for fine-tuning GPCR functions with potential therapeutic implications.

GPCR Activation and Endocytosis Induced by a 2D Material Agonist.

Agonist-induced activation and endocytosis of G protein-coupled receptors (GPCRs) are crucial for a number of physiological and pathological processes. However, tools that are available for probing GPCR endocytosis have been insufficient. Here, we developed a two-dimensional (2D) material agonist by supramolecular self-assembly between an endogenous agonist of κ-opioid receptor (KOR) and 2D molybdenum disulfide. The 2D material agonist has proven to be amenable for eliciting GPCR activation and endocytosis in cells stably expressing KOR rather than in those without KOR expression. Using super-resolution microscopy, we also show that the 2D material agonist colocalizes well with GFP-fused KOR intracellularly. Further, the endocytosed 2D material agonist can selectively produce reactive oxygen species in cells that overly express KOR, as controlled by light irradiation.