Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor

Agonist-induced endocytosis and processing of the G protein-coupled AT1 angiotensin II (Ang II) receptor (AT1R) was studied in HEK 293 cells expressing green fluorescent protein (GFP)- or hemagglutinin epitope-tagged forms of the receptor. After stimulation with Ang II, the receptor and its ligand colocalized with Rab5-GFP and Rab4-GFP in early endosomes, and subsequently with Rab11-GFP in pericentriolar recycling endosomes. Inhibition of phosphatidylinositol (PI) 3-kinase by wortmannin (WT) or LY294002 caused the formation of large endosomal vesicles of heterogeneous Rab composition, containing the ligand-receptor complex in their limiting membranes and in small associated vesicular structures. In contrast to Alexa(R)-transferrin, which was mainly found in small vesicles associated with the outside of large vesicles in WT-treated cells, rhodamine-Ang II was also segregated into small internal vesicles. In cells labeled with 125I-Ang II, WT treatment did not impair the rate of receptor endocytosis, but significantly reduced the initial phase of receptor recycling without affecting its slow component. Similarly, WT inhibited the early, but not the slow, component of the recovery of AT1R at the cell surface after termination of Ang II stimulation. These data indicate that internalized AT1 receptors are processed via vesicles that resemble multivesicular bodies, and recycle to the cell surface by a rapid PI 3-kinase-dependent recycling route, as well as by a slower pathway that is less sensitive to PI 3-kinase inhibitors.     

Assessment of the roles of ordered lipid microdomains in post-endocytic trafficking of glycosyl-phosphatidylinositol-anchored proteins in mammalian fibroblasts

We have used artificial phosphatidylethanolamine-polyethylene glycol (PE-PEG)-anchored proteins, incorporated into living mammalian cells, to evaluate previously proposed roles for ordered lipid 'raft' domains in the post-endocytic trafficking of glycosylphosphatidylinositol (GPI)-anchored proteins in CHO and BHK cells. In CHO cells, endocytosed PE-PEG protein conjugates colocalized strongly with the internalized GPI-anchored folate receptor, concentrating in the endosomal recycling compartment, regardless of the structure of the hydrocarbon chains of the PE-PEG 'anchor'. However, internalized PE-PEG protein conjugates with long-chain saturated anchors recycled to the plasma membrane at a slow rate comparable to that measured for the GPI-anchored folate receptor, whereas conjugates with short-chain or unsaturated anchors recycled at a faster rate similar to that observed for the transferrin receptor. These findings support the proposal (Mayor et al. Cholesterol-dependent retention of GPI-anchored proteins in endosomes. EMBO J 1998;17:4628-4638) that the slow recycling of GPI proteins in CHO cells rests on their affinity for ordered lipid domains. In BHK cells, internalized PE-PEG protein conjugates with either saturated or unsaturated 'anchors' colocalized strongly with simultaneously endocytosed folate receptor and, like the folate receptor, gradually accumulated in late endosomes/lysosomes. These latter findings do not support previous suggestions that the sorting of GPI proteins to late endosomes in BHK cells depends on their association with lipid rafts.     

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of beta2-adrenergic receptors

Phosphatidylinositol 3-kinase inhibitors have been shown to affect endocytosis or subsequent intracellular sorting in various receptor systems. Agonist-activated beta(2)-adrenergic receptors undergo desensitization by mechanisms that include the phosphorylation, endocytosis and degradation of receptors. Following endocytosis, most internalized receptors are sorted to the cell surface, but some proportion is sorted to lysosomes for degradation. It is not known what governs the ratio of receptors that recycle versus receptors that undergo degradation. To determine if phosphatidylinositol 3-kinases regulate beta(2)-adrenergic receptor trafficking, HEK293 cells stably expressing these receptors were treated with the phosphatidylinositol 3-kinase inhibitors LY294002 or wortmannin. We then studied agonist-induced receptor endocytosis and postendocytic sorting, including recycling and degradation of the internalized receptors. Both inhibitors amplified the internalization of receptors after exposure to the beta-agonist isoproterenol, which was attributable to the sorting of a significant fraction of receptors to an intracellular compartment from which receptor recycling did not occur. The initial rate of beta(2)-adrenergic receptor endocytosis and the default rate of receptor recycling were not significantly altered. During prolonged exposure to agonist, LY294002 slowed the degradation rate of beta(2)-adrenergic receptors and caused the accumulation of receptors within rab7-positive vesicles. These results suggest that phosphatidylinositol 3-kinase inhibitors (1) cause a misrouting of beta(2)-adrenergic receptors into vesicles that are neither able to efficiently recycle to the surface nor sort to lysosomes, and (2) delays the movement of receptors from late endosomes to lysosomes.     

Role of the cyclic AMP-dependent protein kinase in homologous resensitization of the beta1-adrenergic receptor

A fundamental question in biology is how the various motifs in G protein-coupled receptors participate in the divergent functions orchestrated by these molecules. Here we describe a fundamental role for a serine residue at position 312 in the third intracellular loop of the human beta(1)-adrenergic receptor (beta(1)-AR) in endocytic recycling of the agonist-internalized receptor. In receptor recycling experiments that were monitored by confocal microscopy, the agonist-internalized wild-type (WT) beta(1)-AR recycled with a t(0.5) of 14 +/- 3 min. Mutagenesis of Ser(312) to alanine (Ser(312) --> Ala beta(1)-AR) or to the phosphoserine mimic aspartic acid (Ser(312) --> Asp beta(1)-AR) resulted in beta(1)-AR constructs that were pharmacologically indistinguishable from the WT beta(1)-AR. The internalized Ser(312) --> Asp beta(1)-AR recycled efficiently with a t(0.5) of 11 +/- 3 min, whereas the internalized Ser(312) --> Ala beta(1)-AR was not recycled or functionally resensitized through the endosomal pathway. Because this serine is a putative residue for phosphorylation by the cyclic AMP-dependent protein kinase (PKA), we examined the role of this kinase in recycling of the internalized beta(1)-AR. Inhibition of PKA biochemically or genetically using a dominant negative PKA construct blocked the recycling of the internalized WT beta(1)-AR. Phosphorylation studies revealed that the beta(1)-AR is partially phosphorylated by PKA and that phosphorylation of the beta(1)-AR by the catalytic subunit of PKA occurs exclusively at Ser(312). Our results identify a new signaling paradigm in which homologous activation of a kinase provides a reversible modification that shifts the itinerary of the internalized receptor toward recycling and resensitization. Therefore, PKA-mediated phosphorylation of G protein-coupled receptors might result in motif-dependent desensitization or resensitization.     

The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions

Receptor recycling involves two endosome populations, peripheral early endosomes and perinuclear recycling endosomes. In polarized epithelial cells, either or both populations must be able to sort apical from basolateral proteins, returning each to its appropriate plasma membrane domain. However, neither the roles of early versus recycling endosomes in polarity nor their relationship to each other has been quantitatively evaluated. Using a combined morphological, biochemical, and kinetic approach, we found these two endosome populations to represent physically and functionally distinct compartments. Early and recycling endosomes were resolved on Optiprep gradients and shown to be differentially associated with rab4, rab11, and transferrin receptor; rab4 was enriched on early endosomes and at least partially depleted from recycling endosomes, with the opposite being true for rab11 and transferrin receptor. The two populations were also pharmacologically distinct, with AlF4 selectively blocking export of transferrin receptor from recycling endosomes to the basolateral plasma membrane. We applied these observations to a detailed kinetic analysis of transferrin and dimeric IgA recycling and transcytosis. The data from these experiments permitted the construction of a testable, mathematical model which enabled a dissection of the roles of early and recycling endosomes in polarized receptor transport. Contrary to expectations, the majority (>65%) of recycling to the basolateral surface is likely to occur from early endosomes, but with relatively little sorting of apical from basolateral proteins. Instead, more complete segregation of basolateral receptors from receptors intended for transcytosis occurred upon delivery to recycling endosomes.     

Structural basis for Rab11-mediated recruitment of FIP3 to recycling endosomes

The Rab11 GTPase regulates recycling of internalized plasma membrane receptors and is essential for completion of cytokinesis. A family of Rab11 interacting proteins (FIPs) that conserve a C-terminal Rab-binding domain (RBD) selectively recognize the active form of Rab11. Normal completion of cytokinesis requires a complex between Rab11 and FIP3. Here, we report the crystal structure and mutational analysis of a heterotetrameric complex between constitutively active Rab11 and a FIP3 construct that includes the RBD. Two Rab11 molecules bind to dyad symmetric sites at the C terminus of FIP3, which forms a non-canonical coiled-coiled dimer with a flared C terminus and hook region. The RBD overlaps with the coiled coil and extends through the C-terminal hook. Although FIP3 engages the switch and interswitch regions of Rab11, the mode of interaction differs significantly from that of other Rab-effector complexes. In particular, the switch II region undergoes a large structural rearrangement from an ordered but non-complementary active conformation to a remodeled conformation that facilitates the interaction with FIP3. Finally, we provide evidence that FIP3 can form homo-oligomers in cells, and that a critical determinant of Rab11 binding in vitro is necessary for FIP3 recruitment to recycling endosomes during cytokinesis.     

Modulation of receptor cycling by neuron-enriched endosomal protein of 21 kD

Although correct cycling of neuronal membrane proteins is essential for neurite outgrowth and synaptic plasticity, neuron-specific proteins of the implicated endosomes have not been characterized. Here we show that a previously cloned, developmentally regulated, neuronal protein of unknown function binds to syntaxin 13. We propose to name this protein neuron-enriched endosomal protein of 21 kD (NEEP21), because it is colocalized with transferrin receptors, internalized transferrin (Tf), and Rab4. In PC12 cells, NEEP21 overexpression accelerates Tf internalization and recycling, whereas its down-regulation strongly delays Tf recycling. In primary neurons, NEEP21 is localized to the somatodendritic compartment, and, upon N-methyl-d-aspartate (NMDA) stimulation, the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor subunit GluR2 is internalized into NEEP21-positive endosomes. NEEP21 down-regulation retards recycling of GluR1 to the cell surface after NMDA stimulation of hippocampal neurons. In summary, NEEP21 is a neuronal protein that is localized to the early endosomal pathway and is necessary for correct receptor recycling in neurons.     

Hypertonia-associated protein Trak1 is a novel regulator of endosome-to-lysosome trafficking

Hypertonia, which is characterized by stiff gait, abnormal posture, jerky movements, and tremor, is associated with a number of neurological disorders, including cerebral palsy, dystonia, Parkinson's disease, stroke, and spinal cord injury. Recently, a spontaneous mutation in the gene encoding trafficking protein, kinesin-binding 1 (Trak1), was identified as the genetic defect that causes hypertonia in mice. The subcellular localization and biological function of Trak1 remain unclear. Here we report that Trak1 interacts with hepatocyte-growth-factor-regulated tyrosine kinase substrate (Hrs), an essential component of the endosomal sorting and trafficking machinery. Double-label immunofluorescence confocal studies show that the endogenous Trak1 protein partially colocalizes with Hrs on early endosomes. Like Hrs, both overexpression and small-interfering-RNA-mediated knockdown of Trak1 inhibit degradation of internalized epidermal growth factor receptors through a block in endosome-to-lysosome trafficking. Our findings support a role for Trak1 in the regulation of Hrs-mediated endosomal sorting and have important implications for understanding hypertonia associated with neurological disorders.     

Transferrin receptor recycling in the absence of perinuclear recycling endosomes

In mammalian cells, internalized receptors such as transferrin (Tfn) receptor are presumed to pass sequentially through early endosomes (EEs) and perinuclear recycling endosomes (REs) before returning to the plasma membrane. Whether passage through RE is obligatory, however, remains unclear. Kinetic analysis of endocytosis in CHO cells suggested that the majority of internalized Tfn bypassed REs returning to the surface from EEs. To determine directly if REs are dispensable for recycling, we studied Tfn recycling in cytoplasts microsurgically created to contain peripheral EEs but to exclude perinuclear REs. The cytoplasts actively internalized and recycled Tfn. Surprisingly, they also exhibited spatially and temporally distinct endosome populations. The first appeared to correspond to EEs, labeling initially with Tfn, being positive for early endosomal antigen 1 (EEA-1) and containing only small amounts of Rab11, an RE marker. The second was EEA-1 negative and with time recruited Rab11, suggesting that cytoplasts assembled functional REs. These results suggest that although perinuclear REs are not essential components of the Tfn recycling pathway, they are dynamic structures which preexist in the peripheral cytoplasm or can be regenerated from EE- and cytosol-derived components such as Rab11.     

SNX1 defines an early endosomal recycling exit for sortilin and mannose 6-phosphate receptors

Mannose-6-phosphate receptors (MPRs) transport lysosomal hydrolases from the trans Golgi network (TGN) to endosomes. Recently, the multi-ligand receptor sortilin has also been implicated in this transport, but the transport carriers involved herein have not been identified. By quantitative immuno-electron microscopy, we localized endogenous sortilin of HepG2 cells predominantly to the TGN and endosomes. In the TGN, sortilin colocalized with MPRs in the same clathrin-coated vesicles. In endosomes, sortilin and MPRs concentrated in sorting nexin 1 (SNX1)-positive buds and vesicles. SNX1 depletion by small interfering RNA resulted in decreased pools of sortilin in the TGN and an increase in lysosomal degradation. These data indicate that sortilin and MPRs recycle to the TGN in SNX1-dependent carriers, which we named endosome-to-TGN transport carriers (ETCs). Notably, ETCs emerge from early endosomes (EE), lack recycling plasma membrane proteins and by three-dimensional electron tomography exhibit unique structural features. Hence, ETCs are distinct from hitherto described EE-derived membranes involved in recycling. Our data emphasize an important role of EEs in recycling to the TGN and indicate that different, specialized exit events occur on the same EE vacuole.     

Role of ßarrestins in bradykinin B2 receptor-mediated signalling

G protein-coupled receptors (GPCRs) can engage multiple pathways to activate ERK1/2 via both G proteins and/or ßarrestin. Receptor recruitment of ßarrestin is also important for GPCR desensitization, internalization and resensitization. Modulation of the receptor/ßarrestin interaction through modification of either component would presumably alter the output generated by receptor activation. Here we examined how ßarrestins regulate bradykinin (BK) B2 receptor (B2R) signalling and desensitization by either truncating ßarrestin1 or ßarrestin2 or by alanine substitution of a serine/threonine cluster in the C-terminal tail of B2R (B2R-4A), conditions which all affect the avidity of the B2R/ßarrestin complex. We first demonstrate that BK-mediated ERK1/2 activation is biphasic containing an early peak (between 2-5 min) followed by sustained activation for at least 60 min. The early but not the sustained phase was predictably affected by inhibition of either Gαq/11 or Gαi/o, whereas loss of ßarrestin2 but not ßarrestin1 resulted in diminished prolonged ERK1/2 activation. ßarrestin2's role was further examined using a truncation mutant with augmented avidity for the agonist-occupied receptor, revealing an increase in both immediate and extended ERK1/2 signalling. We also show that ERK1/2 is recruited to the B2R/ßarrestin complex on endosomes as well as the plasma membrane. Moreover, we investigated ßarrestin's role using the B2R-4A, which is deficient in ßarrestin binding and does not internalize. We show that ERK1/2 signalling downstream of the receptor is entirely G protein-dependent and receptor-mediated intracellular calcium mobilization studies revealed a lack of desensitization. Functionally, the lack of desensitization resulted in increased cell growth and migration compared to the wild-type receptor, which was sensitive to MEK inhibition. These results highlight ßarrestin's crucial role in the maintenance of proper B2R signalling.     

HCMV-Encoded Glycoprotein M (UL100) Interacts with Rab11 Effector Protein FIP4

The envelope of human cytomegalovirus (HCMV) consists of a large number of glycoproteins. The most abundant glycoprotein in the HCMV envelope is the glycoprotein M (UL100), which together with glycoprotein N (UL73) form the gM/gN protein complex. Using yeast two-hybrid screening, we found that the gM carboxy-terminal cytoplasmic tail (gM-CT) interacts with FIP4, a Rab11-GTPase effector protein. Depletion of FIP4 expression in HCMV-infected cells resulted in a decrease in infectious virus production that was also associated with an alteration of the HCMV assembly compartment (AC) phenotype. A similar phenotype was also observed in HCMV-infected cells that expressed dominant negative Rab11(S25N). Recently, it has been shown that FIP4 interactions with Rab11 and additionally with Arf6/Arf5 are important for the vesicular transport of proteins in the endosomal recycling compartment (ERC) and during cytokinesis. Surprisingly, FIP4 interaction with gM-CT limited binding of FIP4 with Arf5/Arf6; however, FIP4 interaction with gM-CT did not prevent recruitment of Rab11 into the ternary complex. These data argued for a contribution of the ERC during cytoplasmic envelopment of HCMV and showed a novel FIP4 function independent of Arf5 or Arf6 activity.     

Syntaxin 13 Mediates Cycling of Plasma Membrane Proteins via Tubulovesicular Recycling Endosomes

Endocytosis-mediated recycling of plasma membrane is a critical vesicle trafficking step important in diverse biological processes. The membrane trafficking decisions and sorting events take place in a series of heterogeneous and highly dynamic organelles, the endosomes. Syntaxin 13, a recently discovered member of the syntaxin family, has been suggested to play a role in mediating endosomal trafficking. To better understand the function of syntaxin 13 we examined its intracellular distribution in nonpolarized cells. By confocal immunofluorescence and electron microscopy, syntaxin 13 is primarily found in tubular early and recycling endosomes, where it colocalizes with transferrin receptor. Additional labeling is also present in endosomal vacuoles, where it is often found in clathrin-coated membrane areas. Furthermore, anti-syntaxin 13 antibody inhibits transferrin receptor recycling in permeabilized PC12 cells. Immunoprecipitation of syntaxin 13 revealed that, in Triton X-100 extracts, syntaxin 13 is present in a complex(es) comprised of βSNAP, VAMP 2/3, and SNAP-25. This complex(es) binds exogenously added αSNAP and NSF and dissociates in the presence of ATP, but not ATPγS. These results support a role for syntaxin 13 in membrane fusion events during the recycling of plasma membrane proteins.     

Rab11 regulates the compartmentalization of early endosomes required for efficient transport from early endosomes to the trans-golgi network

Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20 degrees C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.     

SCF acts in endosomal sorting of the GH receptor

The ubiquitin ligase SCF^TrCP is required for internalisation of the growth hormone receptor (GHR) and acts via a direct interaction with the ubiquitin-dependent endocytosis motif. Details of how the ligase communicates its information to the clathrin-mediated internalisation machinery are unknown. For the EGF receptor, c-Cbl acts both at the cell surface and in endosomes. We hypothesised that SCF^TrCP is required for GHR degradation at both sites. This was tested by truncating GHR after a di-leucine-based internalisation motif (GHR349). This receptor enters the cells via the adapter complex AP2. We show that TrCP acts in an early stage of cargo selection: both TrCP silencing and mutation of the ubiquitin-dependent endocytosis motif force the GHR to recycle between endosomes and the plasma membrane, together with the transferrin receptor. Depletion of Tsg101 (ESCRT-I) has the same effect, while silencing of Hrs (ESCRT-0) prevents GH recycling. GH passes through late endosomal vesicles, marked by Lamp1. Coexpressing GHR and EGFR demonstrates that both receptors use the same route to the lysosomes. We show for the first time that SCF^TrCP is involved in cargo-specific sorting at endosomes and that Tsg101 rather than Hrs might direct the cargo into the ESCRT machinery.     

The kinesin KIF16B mediates apical transcytosis of transferrin receptor in AP‐1B‐deficient epithelia

Polarized epithelial cells take up nutrients from the blood through receptors that are endocytosed and recycle back to the basolateral plasma membrane (PM) utilizing the epithelial‐specific clathrin adaptor AP‐1B. Some native epithelia lack AP‐1B and therefore recycle cognate basolateral receptors to the apical PM, where they carry out important functions for the host organ. Here, we report a novel transcytotic pathway employed by AP‐1B‐deficient epithelia to relocate AP‐1B cargo, such as transferrin receptor (TfR), to the apical PM. Lack of AP‐1B inhibited basolateral recycling of TfR from common recycling endosomes (CRE), the site of function of AP‐1B, and promoted its transfer to apical recycling endosomes (ARE) mediated by the plus‐end kinesin KIF16B and non‐centrosomal microtubules, and its delivery to the apical membrane mediated by the small GTPase rab11a. Hence, our experiments suggest that the apical recycling pathway of epithelial cells is functionally equivalent to the rab11a‐dependent TfR recycling pathway of non‐polarized cells. They define a transcytotic pathway important for the physiology of native AP‐1B‐deficient epithelia and report the first microtubule motor involved in transcytosis.     

Internalization of the TGF-β type I receptor into caveolin-1 and EEA1 double-positive early endosomes

Endocytosis and intracellular sorting of transforming growth factor-β (TGF-β) receptors play an important regulatory role in TGF-β signaling. Two major endocytic pathways, clathrin- and caveolae-mediated endocytosis, have been reported to independently mediate the internalization of TGF-β receptors. In this study, we demonstrate that the clathrin- and caveolae-mediated endocytic pathways can converge during TGF-β receptor endocytic trafficking. By tracking the intracellular dynamics of fluorescently-labeled TGF-β type I receptor (TβRI), we found that after mediating TβRI internalization, certain clathrin-coated vesicles and caveolar vesicles are fused underneath the plasma membrane, forming a novel type of caveolin-1 and clathrin double-positive vesicles. Under the regulation of Rab5, the fused vesicles are targeted to early endosomes and thus deliver the internalized TβRI to the caveolin-1 and EEA1 double-positive early endosomes (caveolin-1-positive early endosomes). We further showed that the caveolin-1-positive early endosomes are positive for Smad3/SARA, Rab11 and Smad7/Smurf2, and may act as a multifunctional device for TGF-β signaling and TGF-β receptor recycling and degradation. Therefore, these findings uncover a novel scenario of endocytosis, the direct fusion of clathrin-coated and caveolae vesicles during TGF-β receptor endocytic trafficking, which leads to the formation of the multifunctional sorting device, caveolin-1-positive early endosomes, for TGF-β receptors.     

A myosin I is involved in membrane recycling from early endosomes

Geometry-based mechanisms have been proposed to account for the sorting of membranes and fluid phase in the endocytic pathway, yet little is known about the involvement of the actin-myosin cytoskeleton. Here, we demonstrate that Dictyostelium discoideum myosin IB functions in the recycling of plasma membrane components from endosomes back to the cell surface. Cells lacking MyoB (myoA(-)/B(-), and myoB(-) cells) and wild-type cells treated with the myosin inhibitor butanedione monoxime accumulated a plasma membrane marker and biotinylated surface proteins on intracellular endocytic vacuoles. An assay based on reversible biotinylation of plasma membrane proteins demonstrated that recycling of membrane components is severely impaired in myoA/B null cells. In addition, MyoB was specifically found on magnetically purified early pinosomes. Using a rapid-freezing cryoelectron microscopy method, we observed an increased number of small vesicles tethered to relatively early endocytic vacuoles in myoA(-)/B(-) cells, but not to later endosomes and lysosomes. This accumulation of vesicles suggests that the defects in membrane recycling result from a disordered morphology of the sorting compartment.     

Rapid recycling of beta-adrenergic receptors is dependent on the actin cytoskeleton and myosin Vb

For the beta(2)-adrenergic receptor (beta(2)AR), published evidence suggests that an intact actin cytoskeleton is required for the endocytosis of receptors and their proper sorting to the rapid recycling pathway. We have characterized the role of the actin cytoskeleton in the regulation of beta(2)AR trafficking in human embryonic kidney 293 (HEK293) cells using two distinct actin filament disrupting compounds, cytochalasin D and latrunculin B (LB). In cells pretreated with either drug, beta(2)AR internalization into transferrin-positive vesicles was not altered but both agents significantly decreased the rate at which beta(2)ARs recycled to the cell surface. In LB-treated cells, nonrecycled beta(2)ARs were localized to early embryonic antigen 1-positive endosomes and also accumulated in the recycling endosome (RE), but only a small fraction of receptors localized to LAMP-positive late endosomes and lysosomes. Treatment with LB also markedly enhanced the inhibitory effect of rab11 overexpression on receptor recycling. Dissociating receptors from actin by expression of the myosin Vb tail fragment resulted in missorting of beta(2)ARs to the RE, while the expression of various CART fragments or the depletion of actinin-4 had no detectable effect on beta(2)AR sorting. These results indicate that the actin cytoskeleton is required for the efficient recycling of beta(2)ARs, a process that likely is dependent on myosin Vb.     

Use of Kaede fusions to visualize recycling of G protein-coupled receptors

The heptahelical G protein-coupled receptors (GPCRs) are internalized following agonist treatment and either recycle rapidly to the plasma membrane or enter the lysosomal degradation pathway. Many conventional GPCR recycling assays suffer from the fact that receptors arriving from the secretory pathway may interfere with recycling receptors. In this study, we introduce a new methodology to study post-endocytotic GPCR trafficking using fusions with the recently cloned Kaede protein. In contrast to the widely used green fluorescent protein, the fluorescence of Kaede can be converted from green to red using ultraviolet irradiation. Our methodology allows to study recycling of GPCRs microscopically in real-time bypassing problems with secretory pathway receptors. Initially, receptors are internalized using an agonist. Fluorescence signals in endosomes are switched, and trafficking of the receptors to the plasma membrane can be easily visualized by monitoring their new fluorescence. Using this methodology, we show that the corticotropin-releasing factor receptor type 1 belongs to the family of recycling GPCRs. Moreover, we demonstrate by fluorescence correlation spectroscopy that Kaede does not oligomerize when fused to membrane proteins, representing an additional advantage of this technique. The Kaede technology may be a powerful tool to study membrane protein trafficking in general.