Agonist-stimulated beta-adrenergic receptor internalization requires dynamic cytoskeletal actin turnover

Stimulation of beta-adrenergic receptors (betaARs) leads to sequential recruitment of beta-arrestin, AP-2 adaptor protein, clathrin, and dynamin to the receptor complex, resulting in endocytosis. Whether a dynamic actin cytoskeleton is required for betaAR endocytosis is not known. In this study, we have used beta(1)- and beta(2) ARs, two ubiquitously expressed members of the betaAR family, to comprehensively evaluate the requirement of the actin cytoskeleton in receptor internalization. The integrity of the actin cytoskeleton was manipulated with the agent latrunculin B (LB) and mutants of cofilin to depolymerize actin filaments. Treatment of cells with LB resulted in dose-dependent depolymerization of the cortical actin cytoskeleton that was associated with significant attenuation in internalization of beta(2)ARs, beta(1)ARs, and mutants of beta(1)ARs that internalize via either clathrin- or caveolin-dependent pathways. Importantly, LB treatment did not inhibit beta-arrestin translocation or dynamin recruitment to the agonist-stimulated receptor. To unequivocally demonstrate the requirement of the actin cytoskeleton for beta(2)AR endocytosis, we used an actin-binding protein cofilin that biochemically depolymerizes and severs actin filaments. Isoproterenol-mediated internalization of beta(2)AR was completely blocked in the presence of wild type cofilin, which could be rescued by a mutant of cofilin that mimics a constitutive phosphorylated state and leads to normal agonist-stimulated beta(2)AR endocytosis. Finally, treatment with jasplakinolide, an inhibitor of actin turnover, resulted in dose-dependent inhibition of beta(2)AR internalization, suggesting that turnover of actin filaments at the receptor complex is required for endocytosis. Taken together, these data demonstrate that intact and functional dynamic actin cytoskeleton is required for normal betaAR internalization.     

Rab11 regulates the recycling of the beta2-adrenergic receptor through a direct interaction

The beta2ARs (beta(2)-adrenergic receptors) undergo ligand-induced internalization into early endosomes, but then are rapidly and efficiently recycled back to the plasma membrane, restoring the numbers of functional cell-surface receptors. Gathering evidence suggests that, during prolonged exposure to agonist, some beta2ARs also utilize a slow recycling pathway through the perinuclear recycling endosomal compartment regulated by the small GTPase Rab11. In the present study, we demonstrate by co-immunoprecipitation studies that there is a beta2AR-Rab11 association in HEK-293 cells (human embryonic kidney cells). We show using purified His(6)-tagged Rab11 protein and beta2AR intracellular domains fused to GST (glutathione transferase) that Rab11 interacts directly with the C-terminal tail of beta2AR, but not with the other intracellular domains of the receptor. Pull-down and immunoprecipitation assays revealed that the beta2AR interacts preferentially with the GDP-bound form of Rab11. Arg(333) and Lys(348) in the C-terminal tail of the beta2AR were identified as crucial determinants for Rab11 binding. A beta2AR construct with these two residues mutated to alanine, beta2AR RK/AA (R333A/K348A), was generated. Analysis of cell-surface receptors by ELISA revealed that the recycling of beta2AR RK/AA was drastically reduced when compared with wild-type beta2AR after agonist washout, following prolonged receptor stimulation. Confocal microscopy demonstrated that the beta2AR RK/AA mutant failed to co-localize with Rab11 and recycle to the plasma membrane, in contrast with the wild-type receptor. To our knowledge, the present study is the first report of a direct interaction between the beta2AR and a Rab GTPase, which is required for the accurate intracellular trafficking of the receptor.     

The recycling and transcytotic pathways for IgG transport by FcRn are distinct and display an inherent polarity

The Fc receptor FcRn traffics immunoglobulin G (IgG) in both directions across polarized epithelial cells that line mucosal surfaces, contributing to host defense. We show that FcRn traffics IgG from either apical or basolateral membranes into the recycling endosome (RE), after which the actin motor myosin Vb and the GTPase Rab25 regulate a sorting step that specifies transcytosis without affecting recycling. Another regulatory component of the RE, Rab11a, is dispensable for transcytosis, but regulates recycling to the basolateral membrane only. None of these proteins affect FcRn trafficking away from lysosomes. Thus, FcRn transcytotic and recycling sorting steps are distinct. These results are consistent with a single structurally and functionally heterogeneous RE compartment that traffics FcRn to both cell surfaces while discriminating between recycling and transcytosis pathways polarized in their direction of transport.     

Beta(2)-adrenergic receptor down-regulation. Evidence for a pathway that does not require endocytosis.

Sustained activation of most G protein-coupled receptors causes a time-dependent reduction of receptor density in intact cells. This phenomenon, known as down-regulation, is believed to depend on a ligand-promoted change of receptor sorting from the default endosome-plasma membrane recycling pathway to the endosome-lysosome degradation pathway. This model is based on previous studies of epidermal growth factor (EGF) receptor degradation and implies that receptors need to be endocytosed to be down-regulated. In stable clones of L cells expressing beta(2)-adrenergic receptors (beta(2)ARs), sustained agonist treatment caused a time-dependant decrease in both beta(2)AR binding sites and immuno-detectable receptor. Blocking beta(2)AR endocytosis with chemical treatments or by expressing a dominant negative mutant of dynamin could not prevent this phenomenon. Specific blockers of the two main intracellular degradation pathways, lysosomal and proteasome-associated, were ineffective in preventing beta(2)AR down-regulation. Further evidence for an endocytosis-independent pathway of beta(2)AR down-regulation was provided by studies in A431 cells, a cell line expressing both endogenous beta(2)AR and EGF receptors. In these cells, inhibition of endocytosis and inactivation of the lysosomal degradation pathway did not block beta(2)AR down-regulation, whereas EGF degradation was inhibited. These data indicate that, contrary to what is currently postulated, receptor endocytosis is not a necessary prerequisite for beta(2)AR down-regulation and that the inactivation of beta(2)ARs, leading to a reduction in binding sites, may occur at the plasma membrane.     

Sequence-dependent sorting of recycling proteins by actin-stabilized endosomal microdomains

The functional consequences of signaling receptor endocytosis are determined by the endosomal sorting of receptors between degradation and recycling pathways. How receptors recycle efficiently, in a sequence-dependent manner that is distinct from bulk membrane recycling, is not known. Here, in live cells, we visualize the sorting of a prototypical sequence-dependent recycling receptor, the beta-2 adrenergic receptor, from bulk recycling proteins and the degrading delta-opioid receptor. Our results reveal a remarkable diversity in recycling routes at the level of individual endosomes, and indicate that sequence-dependent recycling is an active process mediated by distinct endosomal subdomains distinct from those mediating bulk recycling. We identify a specialized subset of tubular microdomains on endosomes, stabilized by a highly localized but dynamic actin machinery, that mediate this sorting, and provide evidence that these actin-stabilized domains provide the physical basis for a two-step kinetic and affinity-based model for protein sorting into the sequence-dependent recycling pathway.     

Myosin Vb mobilizes recycling endosomes and AMPA receptors for postsynaptic plasticity

Learning-related plasticity at excitatory synapses in the mammalian brain requires the trafficking of AMPA receptors and the growth of dendritic spines. However, the mechanisms that couple plasticity stimuli to the trafficking of postsynaptic cargo are poorly understood. Here we demonstrate that myosin Vb (MyoVb), a Ca2+-sensitive motor, conducts spine trafficking during long-term potentiation (LTP) of synaptic strength. Upon activation of NMDA receptors and corresponding Ca2+ influx, MyoVb associates with recycling endosomes (REs), triggering rapid spine recruitment of endosomes and local exocytosis in spines. Disruption of MyoVb or its interaction with the RE adaptor Rab11-FIP2 abolishes LTP-induced exocytosis from REs and prevents both AMPA receptor insertion and spine growth. Furthermore, induction of tight binding of MyoVb to actin using an acute chemical genetic strategy eradicates LTP in hippocampal slices. Thus, Ca2+-activated MyoVb captures and mobilizes REs for AMPA receptor insertion and spine growth, providing a mechanistic link between the induction and expression of postsynaptic plasticity.     

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.     

Endosome biogenesis is controlled by ER and the cytoskeleton at tripartite junctions

The plasma membrane (PM) and its associated cargo are internalized into small vesicles via endocytosis funneling cargo into endosomes. The endosomal system must efficiently deliver cargos, as well as recycle cargo receptors and membrane to maintain homeostasis. In animal cells, endosome trafficking, maturation, and cargo recycling rely on the actin and microtubule cytoskeleton. Microtubules and their associated motor proteins provide the roads on which endosomes move and fuse during cargo sorting and delivery. In addition, highly dynamic assemblies of actin adjust the shape of the endosomal membrane to promote cargo segregation into budding domains allowing for receptor recycling. Recent work has revealed that the endoplasmic reticulum (ER) frequently acts as an intermediary between endosomes and their cytoskeletal regulators via membrane contact sites (MCSs). This review will discuss the factors which form these tripartite junction between the ER, endosomes, and the cytoskeleton as well as their function.     

Myosin Vb is required for trafficking of the cystic fibrosis transmembrane conductance regulator in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) secretion across fluid-transporting epithelia is regulated, in part, by modulating the number of CFTR Cl(-) channels in the plasma membrane by adjusting CFTR endocytosis and recycling. However, the mechanisms that regulate CFTR recycling in airway epithelial cells remain unknown, at least in part, because the recycling itineraries of CFTR in these cells are incompletely understood. In a previous study, we demonstrated that CFTR undergoes trafficking in Rab11a-specific apical recycling endosomes in human airway epithelial cells. Myosin Vb is a plus-end-directed, actin-based mechanoenzyme that facilitates protein trafficking in Rab11a-specific recycling vesicles in several cell model systems. There are no published studies examining the role of myosin Vb in airway epithelial cells. Thus, the goal of this study was to determine whether myosin Vb facilitates CFTR recycling in polarized human airway epithelial cells. Endogenous CFTR formed a complex with endogenous myosin Vb and Rab11a. Silencing myosin Vb by RNA-mediated interference decreased the expression of wild-type CFTR and DeltaF508-CFTR in the apical membrane and decreased CFTR-mediated Cl(-) secretion across polarized human airway epithelial cells. A recombinant tail domain fragment of myosin Vb attenuated the plasma membrane expression of CFTR by arresting CFTR recycling. The dominant-negative effect was dependent on the ability of the myosin Vb tail fragment to interact with Rab11a. Taken together, these data indicate that myosin Vb is required for CFTR recycling in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells.     

The Membrane‐Associated Protein,Supervillin, Accelerates F‐Actin‐Dependent Rapid Integrin Recycling and Cell Motility

In migrating cells, the cytoskeleton coordinates signal transduction and redistribution of transmembrane proteins, including integrins and growth factor receptors. Supervillin is an F‐actin‐ and myosin II‐binding protein that tightly associates with signaling proteins in cholesterol‐rich, ‘lipid raft’ membrane microdomains. We show here that supervillin also can localize with markers for early and sorting endosomes (EE/SE) and with overexpressed components of the Arf6 recycling pathway in the cell periphery. Supervillin tagged with the photoswitchable fluorescent protein, tdEos, moves both into and away from dynamic structures resembling podosomes at the basal cell surface. Rapid integrin recycling from EE/SE is inhibited in supervillin‐knockdown cells, but the rates of integrin endocytosis and recycling from the perinuclear recycling center (PNRC) are unchanged. A lack of synergy between supervillin knockdown and the actin filament barbed‐end inhibitor, cytochalasin D, suggests that both treatments affect actin‐dependent rapid recycling. Supervillin also enhances signaling from the epidermal growth factor receptor (EGFR) to extracellular signal‐regulated kinases (ERKs) 1 and 2 and increases the velocity of cell translocation. These results suggest that supervillin, F‐actin and associated proteins coordinate a rapid, basolateral membrane recycling pathway that contributes to ERK signaling and actin‐based cell motility.     

Membrane glycoprotein M6A promotes mu-opioid receptor endocytosis and facilitates receptor sorting into the recycling pathway

The interaction of mu-opioid receptor (MOPr) with the neuronal membrane glycoprotein M6a is known to facilitate MOPr endocytosis in human embryonic kidney 293 (HEK293) cells. To further study the role of M6a in the post-endocytotic sorting of MOPr, we investigated the agonist-induced co-internalization of MOPr and M6a and protein targeting after internalization in HEK293 cells that co-expressed HA-tagged MOPr and Myc-tagged M6a. We found that M6a, MOPr, and Rab 11, a marker for recycling endosomes, co-localized in endocytotic vesicles, indicating that MOPr and M6a are primarily targeted to recycling endosomes after endocytosis. Furthermore, co-expression of M6a augmented the post-endocytotic sorting of delta-opioid receptors into the recycling pathway, indicating that M6a might have a more general role in opioid receptor post-endocytotic sorting. The enhanced post-endocytotic sorting of MOPr into the recycling pathway was accompanied by a decrease in agonist-induced receptor down-regulation of M6a in co-expressing cells. We tested the physiological relevance of these findings in primary cultures of cortical neurons and found that co-expression of M6a markedly increased the translocation of MOPrs from the plasma membrane to intracellular vesicles at steady state and significantly enhanced both constitutive and agonist-induced receptor endocytosis. In conclusion, our results strongly indicate that M6a modulates MOPr endocytosis and post-endocytotic sorting and has an important role in receptor regulation.     

Arrestin isoforms dictate differential kinetics of A2B adenosine receptor trafficking

Adenosine mediates the activation of adenylyl cyclase via its interaction with specific A(2A) and A(2B) adenosine receptors. Previously, we demonstrated that arrestins are involved in rapid agonist-promoted desensitization of the A(2B) adenosine receptor (A(2B)AR) in HEK293 cells. In the present study, we investigate the role of arrestins in A(2B)AR trafficking. Initial studies demonstrated that HEK293 cells stably expressing arrestin antisense constructs, which reduce endogenous arrestin levels, effectively reduced A(2B)AR internalization. A(2B)AR recycling after agonist-induced endocytosis was also significantly impaired in cells with reduced arrestin levels. Interestingly, while overexpression of arrestin-2 or arrestin-3 rescued A(2B)AR internalization and recycling, arrestin-3 promoted a significantly faster rate of recycling as compared to arrestin-2. The specificity of arrestin interaction with A(2B)ARs was further investigated using arrestins fused to the green fluorescent protein (arr-2-GFP and arr-3-GFP). Both arrestins underwent rapid translocation (<1 min) from the cytosol to the plasma membrane following A(2B)AR activation. However, longer incubations with agonist (>10 min) revealed that arr-2-GFP but not arr-3-GFP colocalized with the A(2B)AR in rab-5 and transferrin receptor containing early endosomes. At later times, the A(2B)AR but not arr-2-GFP was observed in an apparent endocytic recycling compartment. Thus, while arrestin-2 and arrestin-3 mediate agonist-induced A(2B)AR internalization with relative equal potency, arrestin isoform binding dictates the differential kinetics of A(2B)AR recycling and resensitization.     

Trafficking patterns of beta-arrestin and G protein-coupled receptors determined by the kinetics of beta-arrestin deubiquitination

Agonist-dependent internalization of G protein-coupled receptors via clathrin-coated pits is dependent on the adaptor protein beta-arrestin, which interacts with elements of the endocytic machinery such as AP2 and clathrin. For the beta(2)-adrenergic receptor (beta(2)AR) this requires ubiquitination of beta-arrestin by E3 ubiquitin ligase, Mdm2. Based on trafficking patterns and affinity of beta-arrestin, G protein-coupled receptors are categorized into two classes. For class A receptors (e.g. beta(2)AR), which recycle rapidly, beta-arrestin directs the receptors to clathrin-coated pits but does not internalize with them. For class B receptors (e.g. V2 vasopressin receptors), which recycle slowly, beta-arrestin internalizes with the receptor into endosomes. In COS-7 and human embryonic kidney (HEK)-293 cells, stimulation of the beta(2)AR or V2 vasopressin receptor leads, respectively, to transient or stable beta-arrestin ubiquitination. The time course of ubiquitination and deubiquitination of beta-arrestin correlates with its association with and dissociation from each type of receptor. Chimeric receptors, constructed by switching the cytoplasmic tails of the two classes of receptors (beta(2)AR and V2 vasopressin receptors), demonstrate reversal of the patterns of both beta-arrestin trafficking and beta-arrestin ubiquitination. To explore the functional consequences of beta-arrestin ubiquitination we constructed a yellow fluorescent protein-tagged beta-arrestin2-ubiquitin chimera that cannot be deubiquitinated by cellular deubiquitinases. This "permanently ubiquitinated" beta-arrestin did not dissociate from the beta(2)AR but rather internalized with it into endosomes, thus transforming this class A receptor into a class B receptor with respect to its trafficking pattern. Overexpression of this beta-arrestin ubiquitin chimera in HEK-293 cells also results in enhancement of beta(2)AR internalization and degradation. In the presence of N-ethylmaleimide (an inhibitor of deubiquitinating enzymes), coimmunoprecipitation of the receptor and beta-arrestin was increased dramatically, suggesting that deubiquitination of beta-arrestin triggers its dissociation from the receptor. Thus the ubiquitination status of beta-arrestin determines the stability of the receptor-beta-arrestin complex as well as the trafficking pattern of beta-arrestin.     

Live cell imaging of Gs and the beta2-adrenergic receptor demonstrates that both alphas and beta1gamma7 internalize upon stimulation and exhibit similar trafficking patterns that differ from that of the beta2-adrenergic receptor

To visualize and investigate the regulation of the localization patterns of Gs and an associated receptor during cell signaling, we produced functional fluorescent fusion proteins and imaged them in HEK-293 cells. alphas-CFP, with cyan fluorescent protein (CFP) inserted into an internal loop of alphas, localized to the plasma membrane and exhibited similar receptor-mediated activity to that of alphas. Functional fluorescent beta1gamma7 dimers were produced by fusing an amino-terminal yellow fluorescent protein (YFP) fragment to beta1 (YFP-N-beta1) and a carboxyl-terminal YFP fragment to gamma7 (YFP-C-gamma7). When expressed together, YFP-N-beta1 and YFP-C-gamma7 produced fluorescent signals in the plasma membrane that were not seen when the subunits were expressed separately. Isoproterenol stimulation of cells co-expressing alphas-CFP, YFP-N-beta1/YFP-C-gamma7, and the beta2-adrenergic receptor (beta2AR) resulted in internalization of both fluorescent signals from the plasma membrane. Initially, alphas-CFP and YFP-N-beta1/YFP-C-gamma7 stained the cytoplasm diffusely, and subsequently they co-localized on vesicles that exhibited minimal overlap with beta2AR-labeled vesicles. Moreover, internalization of beta2AR-GFP, but not alphas-CFP or YFP-N-beta1/YFP-C-gamma7, was inhibited by a fluorescent dominant negative dynamin 1 mutant, Dyn1(K44A)-mRFP, indicating that the Gs subunits and beta2AR utilize different internalization mechanisms. Subsequent trafficking of the Gs subunits and beta2AR also differed in that vesicles labeled with the Gs subunits exhibited less overlap with RhoB-labeled endosomes and greater overlap with Rab11-labeled endosomes. Because Rab11 regulates traffic through recycling endosomes, co-localization of alphas and beta1gamma7 on these endosomes may indicate a means of recycling specific alphasbetagamma combinations to the plasma membrane.     

Activation of Protein Kinase C α Is Necessary for Sorting the PDGF β-Receptor to Rab4a-dependent Recycling

Previous studies showed that loss of the T-cell protein tyrosine phosphatase (TC-PTP) induces Rab4a-dependent recycling of the platelet-derived growth factor (PDGF) β-receptor in mouse embryonic fibroblasts (MEFs). Here we identify protein kinase C (PKC) α as the critical signaling component that regulates the sorting of the PDGF β-receptor at the early endosomes. Down-regulation of PKC abrogated receptor recycling by preventing the sorting of the activated receptor into EGFP-Rab4a positive domains on the early endosomes. This effect was mimicked by inhibition of PKCα, using myristoylated inhibitory peptides or by knockdown of PKCα with shRNAi. In wt MEFs, short-term preactivation of PKC by PMA caused a ligand-induced PDGF β-receptor recycling that was dependent on Rab4a function. Together, these observations demonstrate that PKC activity is necessary for recycling of ligand-stimulated PDGF β-receptor to occur. The sorting also required Rab4a function as it was prevented by expression of EGFP-Rab4aS22N. Preventing receptor sorting into recycling endosomes increased the rate of receptor degradation, indicating that the sorting of activated receptors at early endosomes directly regulates the duration of receptor signaling. Activation of PKC through the LPA receptor also induced PDGF β-receptor recycling and potentiated the chemotactic response to PDGF-BB. Taken together, our present findings indicate that sorting of PDGF β-receptors on early endosomes is regulated by sequential activation of PKCα and Rab4a and that this sorting step could constitute a point of cross-talk with other receptors.     

A Role of myosin Vb and Rab11-FIP2 in the aquaporin-2 shuttle

Arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells. Its binding to Gs-coupled vasopressin V2 receptors increases cyclic AMP (cAMP) and subsequently elicits the redistribution of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the plasma membrane (AQP2 shuttle), thereby facilitating water reabsorption from primary urine. The AQP2 shuttle is a paradigm for cAMP-dependent exocytic processes. Using sections of rat kidney, the AQP2-expressing cell line CD8, and primary principal cells, we studied the role of the motor protein myosin Vb, its vesicular receptor Rab11, and the myosin Vb- and Rab11-binding protein Rab11-FIP2 in the AQP2 shuttle. Myosin Vb colocalized with AQP2 intracellularly in resting and at the plasma membrane in AVP-treated cells. Rab11 was found on AQP2-bearing vesicles. A dominant-negative myosin Vb tail construct and Rab11-FIP2 lacking the C2 domain (Rab11-FIP2-DeltaC2), which disrupt recycling, caused condensation of AQP2 in a Rab11-positive compartment and abolished the AQP2 shuttle. This effect was dependent on binding of myosin Vb tail and Rab11-FIP2-DeltaC2 to Rab11. In summary, we identified myosin Vb as a motor protein involved in AQP2 recycling and show that myosin Vb- and Rab11-FIP2-dependent recycling of AQP2 is an integral part of the AQP2 shuttle.     

Myosin Vb Is Associated with Plasma Membrane Recycling Systems

Myosin Va is associated with discrete vesicle populations in a number of cell types, but little is known of the function of myosin Vb. Yeast two-hybrid screening of a rabbit parietal cell cDNA library with dominant active Rab11a (Rab11aS20V) identified myosin Vb as an interacting protein for Rab11a, a marker for plasma membrane recycling systems. The isolated clone, corresponding to the carboxyl terminal 60 kDa of the myosin Vb tail, interacted with all members of the Rab11 family (Rab11a, Rab11b, and Rab25). GFP-myosin Vb and endogenous myosin Vb immunoreactivity codistributed with Rab11a in HeLa and Madin-Darby canine kidney (MDCK) cells. As with Rab11a in MDCK cells, the myosin Vb immunoreactivity was dispersed with nocodazole treatment and relocated to the apical corners of cells with taxol treatment. A green fluorescent protein (GFP)-myosin Vb tail chimera overexpressed in HeLa cells retarded transferrin recycling and caused accumulation of transferrin and the transferrin receptor in pericentrosomal vesicles. Expression of the myosin Vb tail chimera in polarized MDCK cells stably expressing the polymeric IgA receptor caused accumulation of basolaterally endocytosed polymeric IgA and the polymeric IgA receptor in the pericentrosomal region. The myosin Vb tail had no effects on transferrin trafficking in polarized MDCK cells. The GFP-myosin Va tail did not colocalize with Rab11a and had no effects on recycling system vesicle distribution in either HeLa or MDCK cells. The results indicate myosin Vb is associated with the plasma membrane recycling system in nonpolarized cells and the apical recycling system in polarized cells. The dominant negative effects of the myosin Vb tail chimera indicate that this unconventional myosin is required for transit out of plasma membrane recycling systems.     

SNX27 mediates retromer tubule entry and endosome-to-plasma membrane trafficking of signalling receptors

Endocytic sorting of signalling receptors between recycling and degradative pathways is a key cellular process controlling the surface complement of receptors and, accordingly, the cell's ability to respond to specific extracellular stimuli. The β2 adrenergic receptor (β2AR) is a prototypical seven-transmembrane signalling receptor that recycles rapidly and efficiently to the plasma membrane after ligand-induced endocytosis. β2AR recycling is dependent on the receptor's carboxy-terminal PDZ ligand and Rab4. This active sorting process is required for functional resensitization of β2AR-mediated signalling. Here we show that sequence-directed sorting occurs at the level of entry into retromer tubules and that retromer tubules are associated with Rab4. Furthermore, we show that sorting nexin 27 (SNX27) serves as an essential adaptor protein linking β2ARs to the retromer tubule. SNX27 does not seem to directly interact with the retromer core complex, but does interact with the retromer-associated Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex. The present results identify a role for retromer in endocytic trafficking of signalling receptors, in regulating a receptor-linked signalling pathway, and in mediating direct endosome-to-plasma membrane traffic.     

beta 2-adrenergic receptor internalization, endosomal sorting, and plasma membrane recycling are regulated by rab GTPases

Rab GTPases are recognized as critical regulatory factors involved in vesicular membrane transport and endosomal fusion. For example, Rab5 directs the transport and fusion of endocytic vesicles to and with early endosomes, whereas Rab4 is thought to control protein trafficking from early endosomes back to the plasma membrane. In the present study, we investigated the role of Rab5 and Rab4 GTPases in regulating the endocytosis, intracellular sorting, and the plasma membrane recycling of the beta(2)AR. In cells expressing the dominant-negative Rab5-S34N mutant, beta(2)AR internalization was impaired, and beta(2)AR-bearing endocytic vesicles remained in either close juxtaposition or physically attached to the plasma membrane. In contrast, a constitutively active Rab5-Q79L mutant redirected internalized beta(2)AR to enlarged endosomes but did not prevent beta(2)AR dephosphorylation and recycling. The expression of either wild-type Rab4 or a Rab4-N121I mutant did not prevent beta(2)AR dephosphorylation. However, the dominant-negative Rab4-N121I mutant blocked beta(2)AR resensitization by blocking receptor recycling from endosomes back to the cell surface. Our data indicate that, in addition to regulating the intracellular trafficking and fusion of beta(2)AR-bearing endocytic vesicles, Rab5 also contributes to the formation and/or budding of clathrin-coated vesicles. Furthermore, beta(2)AR dephosphorylation occurs as the receptor transits between Rab5- and Rab4-positive compartments.     

Rab11-family interacting protein 2 and myosin Vb are required for CXCR2 recycling and receptor-mediated chemotaxis

Agonist-stimulated internalization followed by recycling to the cell membrane play an important role in fine-tuning the activity of chemokine receptors. Because the recycling of chemokine receptors is critical for the reestablishment of the cellular responsiveness to ligand, it is crucial to understand the mechanisms underlying the receptor recycling and resensitization. In the present study, we have demonstrated that the chemokine receptor CXCR2 associated with myosin Vb and Rab11-family interacting protein 2 (FIP2) in a ligand-dependent manner. Truncation of the C-terminal domain of the receptor did not affect the association, suggesting that the interactions occur upstream of the C terminus of CXCR2. After ligand stimulation, the internalized CXCR2 colocalized with myosin Vb and Rab11-FIP2 in Rab11a-positive vesicles. The colocalization lasted for approximately 2 h, and little colocalization was observed after 4 h of ligand stimulation. CXCR2 also colocalized with myosin Vb tail or Rab11-FIP2 (129-512), the N-terminal-truncated mutants of myosin Vb and Rab11-FIP2, respectively, but in a highly condensed manner. Expression of the enhanced green fluorescent protein-tagged myosin Vb tail significantly retarded the recycling and resensitization of CXCR2. CXCR2 recycling was also reduced by the expression Rab11-FIP2 (129-512). Moreover, expression of the myosin Vb tail reduced CXCR2- and CXCR4-mediated chemotaxis. These data indicate that Rab11-FIP2 and myosin Vb regulate CXCR2 recycling and receptor-mediated chemotaxis and that passage of internalized CXCR2 through Rab11a-positive recycling system is critical for physiological response to a chemokine.