Role of the Rab11-associated intracellular pool of receptors formed by constitutive endocytosis of the beta isoform of the thromboxane A2 receptor (TP beta)

Intracellular trafficking pathways of G protein-coupled receptors (GPCRs), following their agonist-induced endocytosis and their consequences on receptor function, are the subject of intense research efforts. However, less is known regarding their constitutive endocytosis. We previously demonstrated that the beta isoform of the thromboxane A(2) receptor (TPbeta) undergoes constitutive and agonist-induced endocytosis. Constitutive endocytosis of GPCRs can lead to the formation of an intracellular pool of receptors from which they can recycle back to the cell surface. In the present report, we show with the help of two TPbeta mutants (TPbeta-Y339A and TPbeta-I343A) specifically deficient in constitutive endocytosis that this intracellular pool of receptors serves to maintain agonist sensitivity over prolonged receptor stimulation in HEK293 cells. Second messenger generation by the TPbeta-Y339A and TPbeta-I343A mutants was drastically reduced compared to the wild-type receptor as suggested by dose-response and time-course experiments of inositol phosphates production following agonist treatment, despite normal coupling between the receptors and the Galpha(q) protein. Moreover, second messenger production after receptor activation was dramatically reduced when cells were pretreated with monensin, a recycling inhibitor. Receptor cell surface expression and endocytosis experiments further revealed that the small GTPase Rab11 protein is a determinant factor in controlling TPbeta recycling back to the cell surface. Co-localization experiments performed by immunofluorescence microscopy indicated that both constitutive and agonist-triggered endocytosis resulted in targeting of TPbeta to the Rab11-positive recycling endosome. Thus, we provide evidence that constitutive endocytosis of TPbeta forms a pool of receptors in the perinuclear recycling endosome from which they recycle to the cell surface, a process involved in preserving receptor sensitivity to agonist stimulation.     

Endocytosis and recycling of muscarinic receptors

Agonist stimulation causes the endocytosis of many G protein-coupled receptors, including muscarinic acetylcholine receptors. In this study we have investigated the agonist-triggered trafficking of the M3 muscarinic receptor expressed in SH-SY5Y human neuroblastoma cells. We have compared the ability of a series of agonists to generate the second messenger Ins(1,4,5)P3 with their ability to stimulate receptor endocytosis. We show that there is a good correlation between the intrinsic activity of the agonists and their ability to increase the rate constant for receptor endocytosis. Furthermore, on the basis of our results, we predict that even very weak partial agonists should under some circumstances be able to cause substantial receptor internalization. Receptor endocytosis occurs too slowly to account for the rapid desensitization of the Ca2+ response to carbachol. Instead, receptor endocytosis and recycling appear to play an important role in resensitization. After an initial agonist challenge, the response to carbachol is fully recovered when only about half of the receptors have been recycled to the cell surface, suggesting that there is a receptor reserve of about 50%. Removal of this reserve by receptor alkylation significantly reduces the extent of resensitization. Resensitization is also reduced by inhibitors of either endocytosis alone (concanavalin A) or of endocytosis and recycling (nigericin). Finally, the protein phosphatase inhibitor calyculin A also reduces resensitization, possibly by blocking the dephosphorylation of the receptors in an endosomal compartment.     

The Na+/H+ exchanger regulatory factor stabilizes epidermal growth factor receptors at the cell surface

Ligand binding to cell surface receptors initiates both signal transduction and endocytosis. Although signaling may continue within the endocytic compartment, down-regulation is the major mechanism that controls the concentration of cell surface receptors, their ability to receive environmental signals, and the ultimate strength of biological signaling. Internalization, recycling, and trafficking of receptor tyrosine kinases (RTKs) within the endosome compartment are each regulated to control the overall process of down-regulation. We have identified the Na(+)/H(+) exchanger regulatory factor (NHERF) as an important molecular component that stabilizes epidermal growth factor receptors (EGFRs) at the cell surface to restrict receptor down-regulation. The NH(2)-terminal PDZ domain (PDZ 1) of NHERF specifically binds to an internal peptide motif located within the COOH-terminal regulatory domain of EGFR. Expression of NHERF slows the rate of EGF-induced receptor degradation. A point mutation that abolishes the PDZ 1 recognition sequence of EGFR enhances the rate of ligand-induced endocytosis and down-regulation of EGFR. Similarly, expression of a dominant negative mutant of NHERF enhances EGF-induced receptor down-regulation. In contrast to beta-adrenergic receptors where NHERF enhances recycling of internalized receptors, NHERF stabilizes EGFR at the cell surface and slows the rate of endocytosis without affecting recycling. Although the mechanisms differ, for both RTKs and G protein-coupled receptors, the overall effect of NHERF is to enhance the fraction of receptors present at the cell surface.     

Identification of a novel endocytic recycling signal in the D1 dopamine receptor

A critical event determining the functional consequences of G protein-coupled receptor (GPCR) endocytosis is the molecular sorting of internalized receptors between divergent recycling and degradative membrane pathways. The D1 dopamine receptor recycles rapidly and efficiently to the plasma membrane after agonist-induced endocytosis and is remarkably resistant to proteolytic down-regulation. Whereas the mechanism mediating agonist-induced endocytosis of D1 receptors has been investigated in some detail, little is known about how receptors are sorted after endocytosis. We have identified a sequence present in the carboxyl-terminal cytoplasmic domain of the human D1 dopamine receptor that is specifically required for the efficient recycling of endocytosed receptors back to the plasma membrane. This sequence is distinct from previously identified membrane trafficking signals and is located in a proximal portion of the carboxyl-terminal cytoplasmic domain, in contrast to previously identified GPCR recycling signals present at the distal tip. Nevertheless, fusion of this sequence to the carboxyl terminus of a chimeric mutant delta opioid neuropeptide receptor is sufficient to re-route internalized receptors from lysosomal to recycling membrane pathways, defining this sequence as a bona fide endocytic recycling signal that can function in both proximal and distal locations. These results identify a novel sorting signal controlling the endocytic trafficking itinerary of a physiologically important dopamine receptor, provide the first example of such a sorting signal functioning in a proximal portion of the carboxyl-terminal cytoplasmic domain, and suggest the existence of a diverse array of sorting signals in the GPCR superfamily that mediate subtype-specific regulation of receptors via endocytic membrane trafficking.     

Cargo-Mediated Regulation of a Rapid Rab4-Dependent Recycling Pathway

Membrane trafficking is well known to regulate receptor-mediated signaling processes, but less is known about whether signaling receptors conversely regulate the membrane trafficking machinery. We investigated this question by focusing on the beta-2 adrenergic receptor (B2AR), a G protein-coupled receptor whose cellular signaling activity is controlled by ligand-induced endocytosis followed by recycling. We used total internal reflection fluorescence microscopy (TIR-FM) and tagging with a pH-sensitive GFP variant to image discrete membrane trafficking events mediating B2AR endo- and exocytosis. Within several minutes after initiating rapid endocytosis of B2ARs by the adrenergic agonist isoproterenol, we observed bright “puffs” of locally increased surface fluorescence intensity representing discrete Rab4-dependent recycling events. These events reached a constant frequency in the continuous presence of isoproterenol, and agonist removal produced a rapid (observed within 1 min) and pronounced (≈twofold) increase in recycling event frequency. This regulation required receptor signaling via the cAMP-dependent protein kinase (PKA) and a specific PKA consensus site located in the carboxyl-terminal cytoplasmic tail of the B2AR itself. B2AR-mediated regulation was not restricted to this membrane cargo, however, as transferrin receptors packaged in the same population of recycling vesicles were similarly affected. In contrast, net recycling measured over a longer time interval (10 to 30 min) was not detectably regulated by B2AR signaling. These results identify rapid regulation of a specific recycling pathway by a signaling receptor cargo.     

Fast modulation of μ-opioid receptor (MOR) recycling is mediated by receptor agonists

The μ-opioid receptor (MOR) is a member of the G protein-coupled receptor family and the main target of endogenous opioid neuropeptides and morphine. Upon activation by ligands, MORs are rapidly internalized via clathrin-coated pits in heterologous cells and dissociated striatal neurons. After initial endocytosis, resensitized receptors recycle back to the cell surface by vesicular delivery for subsequent cycles of activation. MOR trafficking has been linked to opioid tolerance after acute exposure to agonist, but it is also involved in the resensitization process. Several studies describe the regulation and mechanism of MOR endocytosis, but little is known about the recycling of resensitized receptors to the cell surface. To study this process, we induced internalization of MOR with [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) and morphine and imaged in real time single vesicles recycling receptors to the cell surface. We determined single vesicle recycling kinetics and the number of receptors contained in them. Then we demonstrated that rapid vesicular delivery of recycling MORs to the cell surface was mediated by the actin-microtubule cytoskeleton. Recycling was also dependent on Rab4, Rab11, and the Ca(2+)-sensitive motor protein myosin Vb. Finally, we showed that recycling is acutely modulated by the presence of agonists and the levels of cAMP. Our work identifies a novel trafficking mechanism that increases the number of cell surface MORs during acute agonist exposure, effectively reducing the development of opioid tolerance.     

The GIT–PIX complexes regulate the chemotactic response of rat basophilic leukaemia cells

Background information. Cell motility entails the reorganization of the cytoskeleton and membrane trafficking for effective protrusion. The GIT–PIX protein complexes are involved in the regulation of cell motility and adhesion and in the endocytic traffic of members of the family of G‐protein‐coupled receptors. We have investigated the function of the endogenous GIT complexes in the regulation of cell motility stimulated by fMLP (formyl‐Met‐Leu‐Phe) peptide, in a rat basophilic leukaemia RBL‐2H3 cell line stably expressing an HA (haemagglutinin)‐tagged receptor for the fMLP peptide. Results. Our analysis shows that RBL cells stably transfected with the chemoattractant receptor expressed both GIT1–PIX and GIT2–PIX endogenous complexes. We have used silencing of the different members of the complex by small interfering RNAs to study the effects on a number of events linked to agonist‐induced cell migration. We found that cell adhesion was not affected by depletion of any of the proteins of the GIT complex, whereas agonist‐enhanced cell spreading was inhibited. Analysis of agonist‐stimulated haptotactic cell migration indicated a specific positive effect of GIT1 depletion on trans‐well migration. The internalization of the formyl‐peptide receptor was also inhibited by depletion of GIT1 and GIT2. The effects of the GIT complexes on trafficking of the receptors was confirmed by an antibody‐enhanced agonist‐induced internalization assay, showing that depletion of PIX, GIT1 or GIT2 protein caused decreased perinuclear accumulation of internalized receptors. Conclusions. Our results show that endogenous GIT complexes are involved in the regulation of chemoattractant‐induced cell motility and receptor trafficking, and support previous findings indicating an important function of the GIT complexes in the regulation of different G‐protein‐coupled receptors. Our results also indicate that endogenous GIT1 and GIT2 regulate distinct subsets of agonist‐induced responses and suggest a possible functional link between the control of receptor trafficking and the regulation of cell motility by GIT proteins.     

Rab5-dependent trafficking of the m4 muscarinic acetylcholine receptor to the plasma membrane, early endosomes, and multivesicular bodies

The m4 subtype of muscarinic acetylcholine receptor regulates many physiological processes and is a novel therapeutic target for neurologic and psychiatric disorders. However, little is known about m4 regulation because of the lack of pharmacologically selective ligands. A crucial component of G protein-coupled receptor regulation is intracellular trafficking. We thus used subtype-specific antibodies and quantitative immunocytochemistry to characterize the intracellular trafficking of m4. We show that following carbachol stimulation, m4 co-localizes with transferrin, and the selective marker of early endosomes, EEA1. In addition, m4 intracellular localization depends on Rab5 activity. The dominant negative Rab5S34N inhibits m4 endocytosis initially following carbachol stimulation, and reduces the size of m4 containing vesicles. The constitutively active Rab5Q79L enhances m4 intracellular distribution, even in unstimulated cells. Rab5Q79L also produces strikingly enlarged vacuoles, which by electron microscopy contain internal vesicles, suggesting that they are multivesicular bodies. m4 localizes both to the perimeter and interior of these vacuoles. In contrast, transferrin localizes only to the vacuole perimeter, demonstrating divergence of m4 trafficking from the pathway followed by constitutively endocytosed transferrin. We thus suggest a novel model by which multivesicular bodies sort G protein-coupled receptors from a transferrin-positive recycling pathway to a nonrecycling, possibly degradative pathway.     

Changes in G protein-coupled receptor sorting protein affinity regulate postendocytic targeting of G protein-coupled receptors

After activation, most G protein-coupled receptors (GPCRs) are regulated by a cascade of events involving desensitization and endocytosis. Internalized receptors can then be recycled to the plasma membrane, retained in an endosomal compartment, or targeted for degradation. The GPCR-associated sorting protein, GASP, has been shown to preferentially sort a number of native GPCRs to the lysosome for degradation after endocytosis. Here we show that a mutant beta(2) adrenergic receptor and a mutant mu opioid receptor that have previously been described as lacking "recycling signals" due to mutations in their C termini in fact bind to GASP and are targeted for degradation. We also show that a mutant dopamine D1 receptor, which has likewise been described as lacking a recycling signal, does not bind to GASP and is therefore not targeted for degradation. Together, these results indicate that alteration of receptors in their C termini can expose determinants with affinity for GASP binding and consequently target receptors for degradation.     

A transplantable sorting signal that is sufficient to mediate rapid recycling of G protein-coupled receptors

The beta(2)-adrenergic receptor and delta opioid receptor represent distinct G protein-coupled receptors that undergo agonist-induced endocytosis via clathrin-coated pits but differ significantly in their postendocytic sorting between recycling and degradative membrane pathways, respectively. Previous results indicate that a distal portion of the carboxyl-terminal cytoplasmic domain of the beta(2)-adrenergic receptor, which engages in PDZ domain-mediated protein interaction, is required for efficient recycling of receptors after agonist-induced endocytosis. Here we demonstrate that a four-residue sequence (DSLL) comprising the core of this protein interaction domain functions as a transplantable endocytic sorting signal that is sufficient to re-route endocytosed delta opioid receptor into a rapid recycling pathway, to inhibit proteolytic down-regulation of receptors, and to mediate receptor-autonomous sorting of mutant receptors from the wild type allele when co-expressed in the same cells. These observations define a transplantable signal mediating rapid recycling of a heterologous G protein-coupled receptor, and they suggest that rapid recycling of certain membrane proteins does not occur by bulk membrane flow but is instead mediated by a specific endocytic sorting mechanism.     

Binding of filamin to the C-terminal tail of the calcitonin receptor controls recycling

Many G protein-coupled receptors undergo endocytosis, but the mechanisms involved in endocytic sorting and recycling remain to be fully elucidated. We found that the G protein-coupled calcitonin receptor (CTR) undergoes tonic internalization and accumulates within the cell. Using a fluorescence loss in photobleaching assay, we classified these vesicles functionally as recycling vesicles. In a two-hybrid screening, we found that the actin-binding protein filamin interacted with the C-terminal tail of the CTR. The degradation of the receptor was profoundly increased in the absence of filamin or the CTR-filamin interaction. The absence of filamin was also associated with a marked decrease in recycling of the receptor from the endosomes to the cell surface. In contrast, calcitonin-induced inhibition of spontaneous filamin proteolysis was associated with increased recycling of the receptor to the cell surface and decreased degradation of the CTR, suggesting an important role for filamin in the endocytic sorting and recycling of the internalized CTR.     

Receptor trafficking via the perinuclear recycling compartment accompanied by cell division is necessary for permanent neurotensin cell sensitization and leads to chronic mitogen-activated protein kinase activation

Most G protein-coupled receptors are internalized after interaction with their respective ligand, a process that subsequently contributes to cell desensitization, receptor endocytosis, trafficking, and finally cell resensitization. Although cellular mechanisms leading to cell desensitization have been widely studied, those responsible for cell resensitization are still poorly understood. We examined here the traffic of the high affinity neurotensin receptor (NT1 receptor) following prolonged exposure to high agonist concentration. Fluorescence and confocal microscopy of Chinese hamster ovary, human neuroblastoma (CHP 212), and murine neuroblastoma (N1E-115) cells expressing green fluorescent protein-tagged NT1 receptor revealed that under prolonged treatment with saturating concentrations of neurotensin (NT) agonist, NT1 receptor and NT transiently accumulated in the perinuclear recycling compartment (PNRC). During this cellular event, cell surface receptors remained markedly depleted as detected by both confocal microscopy and (125)I-NT binding assays. In dividing cells, we observed that following prolonged NT agonist stimulation, NT1 receptors were removed from the PNRC, accumulated in dispersed vesicles inside the cytoplasm, and subsequently reappeared at the cell surface. This NT binding recovery allowed for constant cell sensitization and led to a chronic activation of mitogen-activated protein kinases p42 and p44. Under these conditions, the constant activation of NT1 receptor generates an oncogenic regulation. These observations support the potent role for neuropeptides, such as NT, in cancer progression.     

Endocytosis of G Protein-Coupled Receptors and Their Ligands: Is There a Role in Metal Trafficking?

The prevalence of metal dysregulation in many neurodegenerative and neurocognitive disorders has compelled many studying such diseases to investigate the mechanisms underlying metal regulation in the central nervous system. Metal homoeostasis is often complex, with sophisticated, multilayered pathways in operation. G protein-coupled receptors are omnipresent on cell membranes and have intriguing mechanisms of endocytosis and trafficking that may be useful in metal homoeostasis. Indeed, many receptors and/or their cognate ligands are able to bind metals, and in many cases metals are considered to have neuromodulatory roles as a result of receptor binding. In this mini-review, we outline the structural and functional aspects of G protein-coupled receptors with a focus on the mechanisms leading to endocytosis and cellular trafficking. We further highlight how this may help in the trafficking of metal ions, notably copper.     

NHERF1 regulates parathyroid hormone receptor membrane retention without affecting recycling

Na/H exchange regulatory factor-1 (NHERF1) is a PDZ protein that regulates trafficking of several G protein-coupled receptors. The phenotype of NHERF1-null mice suggests that the parathyroid hormone (PTH) receptor (PTH1R) is the principal GPCR interacting with NHERF1. The effect of NHERF1 on receptor recycling is unknown. Here, we characterized NHERF1 effects on PTH1R membrane tethering and recycling by radio-ligand binding and recovery after maximal receptor endocytosis. Using Chinese hamster ovary cells expressing the PTH1R, where NHERF1 expression could be induced by tetracycline, NHERF1 inhibited PTH1R endocytosis and delayed PTH1R recycling. NHERF1 also inhibited PTH-induced receptor internalization in MC4 osteoblast cells. Reducing constitutive NHERF1 levels in HEK-293 cells with short hairpin RNA directed against NHERF1 augmented PTH1R endocytosis in response to PTH. Mutagenesis of the PDZ-binding domains or deletion of the MERM domain of NHERF1 demonstrated that both are required for inhibition of endocytosis and recycling. Likewise, an intact COOH-terminal PDZ recognition motif in PTH1R is needed. The effect of NHERF1 on receptor internalization and recycling was not associated with altered receptor expression or binding, activation, or phosphorylation but involved beta-arrestin and dynamin. We conclude that NHERF1 inhibits endocytosis without affecting PTH1R recycling in MC4 and PTH1R-expressing HEK-293 cells. Such an effect may protect against PTH resistance or PTH1R down-regulation in certain cells harboring NHERF1.     

beta-Arrestin-mediated ADP-ribosylation factor 6 activation and beta 2-adrenergic receptor endocytosis

beta-Arrestins are multifunctional adaptor proteins known to regulate internalization of agonist-stimulated G protein-coupled receptors by linking them to endocytic proteins such as clathrin and AP-2. Here we describe a previously unappreciated mechanism by which beta-arrestin orchestrates the process of receptor endocytosis through the activation of ADP-ribosylation factor 6 (ARF6), a small GTP-binding protein. Involvement of ARF6 in the endocytic process is demonstrated by the ability of GTP-binding defective and GTP hydrolysis-deficient mutants to inhibit internalization of the beta(2)-adrenergic receptor. The importance of regulation of ARF6 function is shown by the ability of the ARF GTPase-activating protein GIT1 to inhibit and of the ARF nucleotide exchange factor, ARNO, to enhance receptor endocytosis. Endogenous beta-arrestin is found in complex with ARNO. Upon agonist stimulation of the receptor, beta-arrestin also interacts with the GDP-liganded form of ARF6, thereby facilitating ARNO-promoted GTP loading and activation of the G protein. Thus, the agonist-driven formation of a complex including beta-arrestin, ARNO, and ARF6 provides a molecular mechanism that explains how the agonist-stimulated receptor recruits a small G protein necessary for the endocytic process and controls its activation.     

GIT1 paxillin-binding domain is a four-helix bundle, and it binds to both paxillin LD2 and LD4 motifs

The G protein-coupled receptor kinase-interacting protein 1 (GIT1) is a multidomain protein that plays an important role in cell adhesion, motility, cytoskeletal remodeling, and membrane trafficking. GIT1 mediates the localization of the p21-activated kinase (PAK) and PAK-interactive exchange factor to focal adhesions, and its activation is regulated by the interaction between its C-terminal paxillin-binding domain (PBD) and the LD motifs of paxillin. In this study, we determined the solution structure of rat GIT1 PBD by NMR spectroscopy. The PBD folds into a four-helix bundle, which is structurally similar to the focal adhesion targeting and vinculin tail domains. Previous studies showed that GIT1 interacts with paxillin through the LD4 motif. Here, we demonstrated that in addition to the LD4 motif, the GIT1 PBD can also bind to the paxillin LD2 motif, and both LD2 and LD4 motifs competitively target the same site on the PBD surface. We also revealed that paxillin Ser(272) phosphorylation does not influence GIT1 PBD binding in vitro. These results are in agreement with the notion that phosphorylation of paxillin Ser(272) plays an essential role in regulating focal adhesion turnover.     

Clathrin-dependent mechanisms of G protein-coupled receptor endocytosis

The heptahelical G protein-coupled receptor (GPCR) family includes approximately 900 members and is the largest family of signaling receptors encoded in the mammalian genome. G protein-coupled receptors elicit cellular responses to diverse extracellular stimuli at the plasma membrane and some internalized receptors continue to signal from intracellular compartments. In addition to rapid desensitization, receptor trafficking is critical for regulation of the temporal and spatial aspects of GPCR signaling. Indeed, GPCR internalization functions to control signal termination and propagation as well as receptor resensitization. Our knowledge of the mechanisms that regulate mammalian GPCR endocytosis is based predominantly on arrestin regulation of receptors through a clathrin- and dynamin-dependent pathway. However, multiple clathrin adaptors, which recognize distinct endocytic signals, are now known to function in clathrin-mediated endocytosis of diverse cargo. Given the vast number and diversity of GPCRs, the complexity of clathrin-mediated endocytosis and the discovery of multiple clathrin adaptors, a single universal mechanism controlling endocytosis of all mammalian GPCRs is unlikely. Indeed, several recent studies now suggest that endocytosis of different GPCRs is regulated by distinct mechanisms and clathrin adaptors. In this review, we discuss the diverse mechanisms that regulate clathrin-dependent GPCR endocytosis.     

Dysbindin Promotes the Post-Endocytic Sorting of G Protein-Coupled Receptors to Lysosomes

Background

Dysbindin, a cytoplasmic protein long known to function in the biogenesis of specialized lysosome-related organelles (LROs), has been reported to reduce surface expression of D2 dopamine receptors in neurons. Dysbindin is broadly expressed, and dopamine receptors are members of the large family of G protein-coupled receptors (GPCRs) that function in diverse cell types. Thus we asked if dysbindin regulates receptor number in non-neural cells, and further investigated the cellular basis of this regulation.

Methodology/Principal Findings

We used RNA interference to deplete endogenous dysbindin in HEK293 and HeLa cells, then used immunochemical and biochemical methods to assess expression and endocytic trafficking of epitope-tagged GPCRs. Dysbindin knockdown up-regulated surface expression of D2 receptors compared to D1 receptors, as reported previously in neurons. This regulation was not mediated by a change in D2 receptor endocytosis. Instead, dysbindin knockdown specifically reduced the subsequent trafficking of internalized D2 receptors to lysosomes. This distinct post-endocytic sorting function explained the minimal effect of dysbindin depletion on D1 receptors, which recycle efficiently and traverse the lysosomal pathway to only a small degree. Moreover, dysbindin regulated the delta opioid receptor, a more distantly related GPCR that is also sorted to lysosomes after endocytosis. Dysbindin was not required for lysosomal trafficking of all signaling receptors, however, as its depletion did not detectably affect down-regulation of the EGF receptor tyrosine kinase. Dysbindin co-immunoprecipitated with GASP-1 (or GPRASP-1), a cytoplasmic protein shown previously to modulate lysosomal trafficking of D2 dopamine and delta opioid receptors by direct interaction, and with HRS that is a core component of the conserved ESCRT machinery mediating lysosome biogenesis and sorting.

Conclusions/Significance

These results identify a distinct, and potentially widespread function of dysbindin in promoting the sorting of specific GPCRs to lysosomes after endocytosis.     

Rapid internalization and recycling of the human neuropeptide Y Y(1) receptor.

Desensitization of G protein-coupled receptors (GPCRs) involves receptor phosphorylation and reduction in the number of receptors at the cell surface. The neuropeptide Y (NPY) Y(1) receptor undergoes fast desensitization. We examined agonist-induced signaling and internalization using NPY Y(1) receptors fused to green fluorescent protein (EGFP). When expressed in HEK293 cells, EGFP-hNPY Y(1) receptors were localized at the plasma membrane, desensitized rapidly as assessed using calcium responses, and had similar properties compared to hNPY Y(1) receptors. Upon agonist challenge, the EGFP signal decreased rapidly (t(1/2) = 107 +/- 3 s) followed by a slow recovery. This decrease was blocked by BIBP3226, a Y(1) receptor antagonist, or by pertussis toxin, in agreement with Y(1) receptor activation. Internalization of EGFP-hNPY Y(1) receptors to acidic endosomal compartments likely accounts for the decrease in the EGFP signal, being absent after pretreatment with monensin. Concanavalin A and hypertonic sucrose, which inhibit clathrin-mediated endocytosis, blocked the decrease in fluorescence. After agonist, intracellular EGFP signals were punctate and co-localized with transferrin-Texas Red, a marker of clathrin-associated internalization and recycling, but not with LysoTracker Red, a lysosomal pathway marker, supporting receptor trafficking to recycling endosomes rather than the late endosomal/lysosomal pathway. Pulse-chase experiments revealed no receptor degradation after internalization. The slow recovery of fluorescence was unaffected by cycloheximide or actinomycin D, indicating that de novo synthesis of receptors was not limiting. Use of a multicompartment model to fit our fluorescence data allows simultaneous determination of internalization and recycling rate constants. We propose that rapid internalization of receptors via the clathrin-coated pits recycling pathway may largely account for the rapid desensitization of NPY Y(1) receptors.