Structure of Reelin repeat 8 and the adjacent C-terminal region

Neuronal development and function are dependent in part on the several roles of the secreted glycoprotein Reelin. Endogenous proteases process this 400 kDa, modular protein, yielding N-terminal, central, and C-terminal fragments that each have distinct roles in Reelin’s function and regulation. The C-terminal fragment comprises Reelin repeat (RR) domains seven and eight, as well as a basic stretch of 32 amino acid residues termed the C-terminal region (CTR), influences Reelin signaling intensity, and has been reported to bind to Neuropilin-1, which serves as a co-receptor in the canonical Reelin signaling pathway. Here, we present a crystal structure of RR8 at 3.0 Å resolution. Analytical ultracentrifugation and small-angle x-ray scattering confirmed that RR8 is monomeric and enabled us to identify the CTR as a flexible, yet compact subdomain. We conducted structurally informed protein engineering to design a chimeric RR8 construct guided by the structural similarities with RR6. Experimental results support a mode of Reelin-receptor interaction reliant on the multiple interfaces coordinating the binding event. Structurally, RR8 resembles other individual RRs, but its structure does show discrete differences that may account for Reelin receptor specificity toward RR6.     

Identification of two distinct structural motifs that, when added to the C-terminal tail of the rat LH receptor, redirect the internalized hormone-receptor complex from a degradation to a recycling pathway

We show that most of the internalized rat LH receptor is routed to a lysosomal degradation pathway whereas a substantial portion of the human LH receptor is routed to a recycling pathway. Chimeras of these two receptors identified a linear amino acid sequence (GTALL) present near the C terminus of the human LH receptor that, when grafted onto the rat LH receptor, redirects most of the rat LH receptor to a recycling pathway. Removal of the GTALL sequence from the human LH receptor failed to affect its routing, however. The GTALL sequence shows homology with the C-terminal tetrapeptide (DSLL) of the beta2-adrenergic receptor, a motif that has been reported to mediate the recycling of the internalized beta2-adrenergic receptor by binding to ezrin-radixin-moesin-binding phosphoprotein-50. Addition of the DSLL tetrapeptide to the C terminus of the rat LH receptor also redirects most of the internalized rat LH receptor to a recycling pathway but, like the recycling of the human LH receptor, this rerouting is not mediated by ezrin-radixin-moesin-binding phosphoprotein-50. We conclude that most of the internalized rat LH receptor is degraded because its C-terminal tail lacks motifs that promote recycling and that two distinct, but homologous, motifs (DSLL at the C terminus or GTALL near the C terminus) can reroute the internalized rat LH receptor to a recycling pathway that is independent of ezrin-radixin-moesin-binding phosphoprotein-50.     

Recycling of the yeast a-factor receptor

The yeast a-factor receptor (Ste3p) is subject to two mechanistically distinct modes of endocytosis: a constitutive, ligand-independent pathway and a ligand-dependent uptake pathway. Whereas the constitutive pathway leads to degradation of the receptor in the vacuole, the present work finds that receptor internalized via the ligand-dependent pathway recycles. With the a-factor ligand continuously present in the culture medium, trafficking of the receptor achieves an equilibrium in which continuing uptake to endosomal compartments is balanced by its recycling return to the plasma membrane. Withdrawal of ligand from the medium leads to a net return of the internalized receptor back to the plasma membrane. Although recycling is demonstrated for receptors that lack the signal for constitutive endocytosis, evidence is provided indicating a participation of recycling in wild-type Ste3p trafficking as well: a-factor treatment both slows wild-type receptor turnover and results in receptor redistribution to intracellular endosomal compartments. Apparently, a-factor acts as a switch, diverting receptor from vacuole-directed endocytosis and degradation, to recycling. A model is presented for how the two Ste3p endocytic modes may collaborate to generate the polarized receptor distribution characteristic of mating cells.     

A Single Mutation at Lysine 241 Alters Expression and Trafficking of the D2 Dopamine Receptor

Ubiquitination of G protein-coupled receptors has been identified to regulate receptor signal transduction including agonist-induced internalization and sorting of internalized receptor for degradation or for recycling. Using co-immunoprecipitation and immunoblot analysis, I found that the membrane-associated D₂ dopamine receptor (DAR) is mono-ubiquitinated in the absence of an agonist following heterologous expression in human embryonic kidney cells (HEK293). By using site-directed mutagenesis, this report shows that the loss of lysine-241, K241A D₂ DAR reduced the amount of membrane-associated D₂ DAR. It is of interest that the K241A D₂ DAR also had a distinctly different ubiquitination pattern than the wild-type D₂ DAR. It is important to note that the ubiquitinated mutant D₂ DAR was degraded through ubiquitin-proteasome pathway. These data provide the factual evidence that a loss of lysine-241 of the D₂ DAR affects receptor ubiquitination and renders the protein susceptible to the proteasomal degradation.     

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.     

Postendocytic Sorting of Constitutively Internalized Dopamine Transporter in Cell Lines and Dopaminergic Neurons

The dopamine transporter (DAT) mediates reuptake of released dopamine and is the target for psychostimulants, such as cocaine and amphetamine. DAT undergoes marked constitutive endocytosis, but little is known about the fate and sorting of the endocytosed transporter. To study DAT sorting in cells lines, we fused the one-transmembrane segment protein Tac to DAT, thereby generating a transporter (TacDAT) with an extracellular antibody epitope suited for trafficking studies. TacDAT was functional and endocytosed constitutively in HEK293 cells. According to an ELISA-based assay, TacDAT intracellular accumulation was increased by the lysosomal protease inhibitor leupeptin and by monensin, an inhibitor of lysosomal degradation and recycling. Monensin also reduced TacDAT surface expression consistent with partial recycling. In both HEK293 cells and in the dopaminergic cell line 1Rb3An27, constitutively internalized TacDAT displayed primary co-localization with the late endosomal marker Rab7, less co-localization with the “short loop” recycling marker Rab4, and little co-localization with the marker of “long loop” recycling endosomes, Rab11. Removal by mutation of N-terminal ubiquitination sites did not affect this sorting pattern. The sorting pattern was distinct from a bona fide recycling membrane protein, the β₂-adrenergic receptor, that co-localized primarily with Rab11 and Rab4. Constitutively internalized wild type DAT probed with the fluorescently tagged cocaine analogue JHC 1-64, exhibited the same co-localization pattern as TacDAT in 1Rb3An27 cells and in cultured midbrain dopaminergic neurons. We conclude that DAT is constitutively internalized and sorted in a ubiquitination-independent manner to late endosomes/lysosomes and in part to a Rab4 positive short loop recycling pathway.     

Reelin is a ligand for lipoprotein receptors

A signaling pathway involving the extracellular protein Reelin and the intracellular adaptor protein Disabled-1 (Dab1) controls cell positioning during mammalian brain development. Here, we demonstrate that Reelin binds directly to lipoprotein receptors, preferably the very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). Binding requires calcium, and it is inhibited in the presence of apoE. Furthermore, the CR-50 monoclonal antibody, which inhibits Reelin function, blocks the association of Reelin with VLDLR. After binding to VLDLR on the cell surface, Reelin is internalized into vesicles. In dissociated neurons, apoE reduces the level of Reelin-induced tyrosine phosphorylation of Dab1. These data suggest that Reelin directs neuronal migration by binding to VLDLR and ApoER2.     

Contribution of a tyrosine-based motif to cellular trafficking of wild-type and truncated NPY Y1 receptors

The human NPY Y₁ receptor undergoes fast agonist-induced internalization via clathrin-coated pits then recycles back to the cell membrane. In an attempt to identify the molecular determinants involved in this process, we studied several C-terminal truncation mutants tagged with EFGP. In the absence of agonist, Y₁ receptors lacking the last 32 C-terminal amino acids (Y₁Δ32) are constitutively internalized, unlike full-length Y₁ receptors. At steady state, internalized Y₁Δ32 receptors co-localize with transferrin, a marker of early and recycling endosomes. Inhibition of constitutive internalization of Y₁Δ32 receptors by hypertonic sucrose or by co-expression of Rab5aS34N, a dominant negative form of the small GTPase Rab5a or depletion of all three isoforms of Rab5 indicates the involvement of clathrin-coated pits. In contrast, a truncated receptor lacking the last 42 C-terminal amino acids (Y₁Δ42) does not constitutively internalize, consistent with the possibility that there is a molecular determinant responsible for constitutive internalization located in the last 10 amino acids of Y₁Δ32 receptors. We show that the agonist-independent internalization of Y₁Δ32 receptors involves a tyrosine-based motif YXXΦ. The potential role of this motif in the behaviour of full-length Y₁ receptors has also been explored. Our results indicate that a C-terminal tyrosine-based motif is critical for the constitutive internalization of truncated Y₁Δ32 receptors. We suggest that this motif is masked in full-length Y₁ receptors which do not constitutively internalize in the absence of agonist.     

The recycling of apolipoprotein E and its amino-terminal 22 kDa fragment: evidence for multiple redundant pathways

A portion of apolipoprotein E (apoE) internalized by hepatocytes is spared degradation and is recycled. To investigate the intracellular routing of recycling apoE, primary hepatocyte cultures from LDL receptor-deficient mice and mice deficient in receptor-associated protein [a model of depressed expression of LDL receptor-related protein (LRP)] were incubated with human VLDL containing 125I-labeled human recombinant apoE3. Approximately 30% of the internalized intact apoE was recycled after 4 h. The N-terminal 22 kDa fragment of apoE was also resecreted, demonstrating that this apoE domain contains sufficient sequence to recycle. The 22 kDa fragment has reduced affinity for lipoproteins, suggesting that apoE recycling is linked to the ability of apoE to bind directly to a recycling receptor. Finally, apoE was found to recycle equally well in the presence of brefeldin A, a drug that blocks transport from the endoplasmic reticulum and leads to collapse of the Golgi stacks. Our studies demonstrate that apoE recycling occurs 1) in the absence of the LDL receptor or under conditions of markedly reduced LRP expression; 2) when apoE lacks the carboxyl-terminal domain, which allows binding to the lipoprotein; and 3) in the absence of an intact Golgi apparatus. We conclude that apoE recycling occurs through multiple redundant pathways.     

β1-Adrenergic Receptor Recycles Via a Membranous Organelle, Recycling Endosome, by Binding with Sorting Nexin27

In cardiomyocytes, β₁-adrenergic receptor (β₁-AR) plays an important role in regulating cardiac functions. Upon continuous ligand stimulation, β₁-AR is internalized and mostly recycled back to the plasma membrane (PM). The recycling endosome (RE) is one of the membranous organelles involved in the protein recycling pathway. To determine whether RE is involved in the internalization of β₁-AR upon ligand stimulation, we evaluated the localization of β₁-AR after stimulation with a β-agonist, isoproterenol (Iso), in β₁-AR-transfected COS-1 cells. After 30 min of Iso treatment and cell surface labeling with the appropriate antibodies, β₁-AR was internalized from PM and translocated into the perinuclear region, the same location as the transferrin receptor, an RE marker. We then evaluated whether sorting nexin 27 (SNX27) participated in the β₁-AR recycling pathway. When β₁-AR and SNX27 were coexpressed, β₁-AR coimmunoprecipitated with SNX27. In addition, shRNA-mediated silencing of SNX27 compromised β₁-AR recycling and enhanced its delivery into lysosome. Overall, β₁-AR on PM was internalized into RE upon Iso stimulation and recycled by RE through binding with SNX27 in COS-1 cells.     

Internalization of the Human Nicotinic Acid Receptor GPR109A Is Regulated by Gi,GRK2, and Arrestin3

Nicotinic acid (niacin) has been widely used as a favorable lipid-lowering drug for several decades, and the orphan G protein-coupled receptor GPR109A has been identified to be a receptor for niacin. Mechanistic investigations have shown that as a G_i-coupled receptor, GPR109A inhibits adenylate cyclase activity upon niacin activation, thereby inhibiting free fatty acid liberation. However, the underlying molecular mechanisms that regulate signaling and internalization of GPR109A remain largely unknown. To further characterize GPR109A internalization, we made a construct to express GPR109A fused with enhanced green fluorescent protein (EGFP) at its carboxyl-terminal end. In stable GPR109A-EGFP-expressing HEK-293 cells, GPR109A-EGFP was mainly localized at the plasma membrane and was rapidly internalized in a dose- and time-dependent manner upon agonist stimulation. GPR109A internalization was completely blocked by hypertonic sucrose, indicating that GPR109A internalizes via the clathrin-coated pit pathway. Further investigation demonstrated that internalized GPR109A was recycled to the cell surface after the removal of agonist, and recycling of the internalized receptors was not blocked by treatment with acidotropic agents, NH₄Cl and monensin. Pertussis toxin pretreatment not only inhibited forskolin-induced cAMP accumulation and intracellular Ca²⁺ mobilization; it also significantly attenuated agonist-promoted GPR109A internalization. Moreover, RNA interference experiments showed that knockdown of GRK2 (G protein-coupled receptor kinase 2) and arrestin3 expression significantly impaired receptor internalization. Taken together, these results indicate that the agonist-induced internalization of GPR109A receptors is regulated by GRK2 and arrestin3 in a pertussis toxin-sensitive manner and that internalized receptor recycling is independent of endosomal acidification.     

Disabled1 regulates the intracellular trafficking of reelin receptors

Reelin is a huge secreted protein that controls proper laminar formation in the developing brain. It is generally believed that tyrosine phosphorylation of Disabled1 (Dab1) by Src family tyrosine kinases is the most critical downstream event in Reelin signaling. The receptors for Reelin belong to the low density lipoprotein receptor family, most of whose members undergo regulated intracellular trafficking. In this study, we propose novel roles for Dab1 in Reelin signaling. We first demonstrated that cell surface expression of Reelin receptors was decreased in Dab1-deficient neurons. In heterologous cells, Dab1 enhanced cell surface expression of Reelin receptors, and this effect was mediated by direct interaction with the receptors. Moreover, Dab1 did not stably associate with the receptors at the plasma membrane in the resting state. When Reelin was added to primary cortical neurons, Dab1 was recruited to the receptors, and its tyrosine residues were phosphorylated. Although Reelin and Dab1 colocalized well shortly after the addition of Reelin, Dab1 was no longer associated with internalized Reelin. When Src family tyrosine kinases were inhibited, internalization of Reelin was severely abrogated, and Reelin colocalized with Dab1 near the plasma membrane for a prolonged period. Taken together, these results indicate that Dab1 regulates both cell surface expression and internalization of Reelin receptors, and these regulations may play a role in correct laminar formation in the developing brain.     

Mechanism and significance of specific proteolytic cleavage of Reelin

Reelin is a secreted glycoprotein essential for normal brain development and function. In the extracellular milieu, Reelin is subject to specific cleavage at two (N-t and C-t) sites. The N-t cleavage of Reelin is implicated in psychiatric and Alzheimer’s diseases, but the molecular mechanism and physiological significance of this cleavage are not completely understood. Particularly, whether the N-t cleavage affects the signaling activity of Reelin remains controversial.Here, we show that the protease in charge of the N-t cleavage of Reelin requires the activity of certain proprotein convertase family for maturation and has strong affinity for heparin. By taking advantage of these observations, we for the first time succeeded in obtaining “Uncleaved” and “Completely Cleaved” Reelin proteins. The N-t cleavage splits Reelin into two distinct fragments and virtually abolishes its signaling activity. These findings provide an important biochemical basis for the function of Reelin proteolysis in brain development and function.     

Rab11a and its binding partners regulate the recycling of the ß1-adrenergic receptor

ß1-adrenergic receptors (ß1-AR) are internalized in response to agonists and then recycle back for another round of signaling. The serine 312 to alanine mutant of the ß1-AR (S312A) is internalized but does not recycle. We determined that WT ß1-AR and S312A were internalized initially to an early sorting compartment because they colocalized by > 70% with the early endosomal markers rab5a and early endosomal antigen-1 (EEA1). Subsequently, the WT ß1-AR trafficked via rab4a-expressing sorting endosomes to recycling endosomes. In recycling endosomes WT ß1-AR were colocalized by > 70% with the rab11 GTPase. S312A did not colocalize with either rab4a or rab11, instead they exited from early endosomes to late endosomes/lysosomes in which they were degraded. Rab11a played a prominent role in recycling of the WT ß1-AR because dominant negative rab11a inhibited, while constitutively active rab11a accelerated the recycling of the ß1-AR. Next, we determined the effect of each of the rab11-interacting proteins on trafficking of the WT ß1-AR. The recycling of the ß1-AR was markedly inhibited when myosin Vb, FIP2, FIP3 and rabphillin were knocked down. These data indicate that rab11a and a select group of its binding partners play a prominent role in recycling of the human ß1-AR.     

Lysophosphatidylcholine as an effector of fatty acid-induced insulin resistance

The mechanism of FFA-induced insulin resistance is not fully understood. We have searched for effector molecules(s) in FFA-induced insulin resistance. Palmitic acid (PA) but not oleic acid (OA) induced insulin resistance in L6 myotubes through C-Jun N-terminal kinase (JNK) and insulin receptor substrate 1 (IRS-1) Ser307 phosphorylation. Inhibitors of ceramide synthesis did not block insulin resistance by PA. However, inhibition of the conversion of PA to lysophosphatidylcholine (LPC) by calcium-independent phospholipase A₂ (iPLA₂) inhibitors, such as bromoenol lactone (BEL) or palmitoyl trifluoromethyl ketone (PACOCF₃), prevented insulin resistance by PA. iPLA₂ inhibitors or iPLA₂ small interfering RNA (siRNA) attenuated JNK or IRS-1 Ser307 phosphorylation by PA. PA treatment increased LPC content, which was reversed by iPLA₂ inhibitors or iPLA₂ siRNA. The intracellular DAG level was increased by iPLA₂ inhibitors, despite ameliorated insulin resistance. Pertussis toxin (PTX), which inhibits LPC action through the G-protein coupled receptor (GPCR)/Gα_i, reversed insulin resistance by PA. BEL administration ameliorated insulin resistance and diabetes in db/db mice. JNK and IRS-1Ser307 phosphorylation in the liver and muscle of db/db mice was attenuated by BEL. LPC content was increased in the liver and muscle of db/db mice, which was suppressed by BEL. These findings implicate LPC as an important lipid intermediate that links saturated fatty acids to insulin resistance.     

The extremely conserved C-terminal region of Reelin is not necessary for secretion but is required for efficient activation of downstream signaling

Reelin is a very large secreted glycoprotein essential for correct development of the mammalian brain. It is also implicated in higher functions and diseases of human brain. However, whether or not secretion of Reelin is regulated and how Reelin transmits signals remain largely unknown. Reelin protein is composed of an N-terminal F-spondin-like domain, Reelin repeats, and a short and highly basic C-terminal region (CTR). The primary sequence of CTR is almost completely conserved among vertebrates except fishes, indicating its importance. A prevailing idea regarding the function of CTR is that it is required for the secretion of Reelin, although this remains unproven. Here we aimed to clarify the function of Reelin CTR. Neither deleting most of CTR nor replacing CTR with unrelated amino acids affected secretion efficiency, indicating that CTR is not absolutely required for the secretion of Reelin. We also found that Reelin mutants without CTR were less potent in activating the downstream signaling in cortical neurons. Although these mutants were able to bind to the Reelin receptor ectodomain as efficiently as wild-type Reelin, quite interestingly, their ability to bind to the isolated cell membrane bearing Reelin receptors or receptor-expressing cells (including cortical neurons) was much weaker than that of wild-type Reelin. Therefore, it is concluded that the CTR of Reelin is not essential for its secretion but is required for efficient activation of downstream signaling events, presumably via binding to an unidentified "co-receptor" molecule(s) on the cell membrane.     

The long and the short cycle. Alternative intracellular routes for trafficking of G-protein-coupled receptors

The C terminus of the human V2 vasopressin receptor contains multiple phosphorylation sites including a cluster of amino acids that when phosphorylated prevents the return of the internalized receptor to the cell surface. To identify the step where the recycling process was interrupted, the trafficking of the V2 receptor was compared with that of the recycling V1a receptor after exposure to ligand. Initially, both receptors internalized in small peripheral endosomes, but a physical separation of their endocytic pathways was subsequently detected. The V1a receptor remained evenly distributed throughout the cytosol, whereas the V2 receptor accumulated in a large aggregation of vesicles in the proximity of the nucleus where it colocalized with the transferrin receptor and Rab11, a small GTP-binding protein that is concentrated in the perinuclear recycling compartment; only marginal colocalization of Rab11 with the V1a receptor was observed. Thus, the V2 receptor was sequestered in the perinuclear recycling compartment. Targeting to the perinuclear recycling compartment was determined by the receptor subtype and not by the inability to recycle, since the mutation S363A in the phosphorylation-dependent retention signal generated a V2 receptor that was recycled via the same compartment. The perinuclear recycling compartment was enriched in beta-arrestin after internalization of either wild type V2 receptor or its recycling mutant, indicating that long term interaction between the receptors and arrestin was not responsible for the intracellular retention. Thus, the fully phosphorylated retention domain overrides the natural tendency of the V2 receptor to recycle and, by preventing its exit from the perinuclear recycling compartment, interrupts its transit via the "long cycle." The data suggest that the inactivation of the domain, possibly by dephosphorylation, triggers the return of the receptor from the perinuclear compartment to the plasma membrane.     

A secreted soluble form of ApoE receptor 2 acts as a dominant-negative receptor and inhibits Reelin signaling

Specialized neurons throughout the developing central nervous system secrete Reelin, which binds to ApoE receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR), triggering a signal cascade that guides neurons to their correct position. Binding of Reelin to ApoER2 and VLDLR induces phosphorylation of Dab1, which binds to the intracellular domains of both receptors. Due to differential splicing, several isoforms of ApoER2 differing in their ligand-binding and intracellular domains exist. One isoform harbors four binding repeats plus an adjacent short 13 amino acid insertion containing a furin cleavage site. It is not known whether furin processing of this ApoER2 variant actually takes place and, if so, whether the produced fragment is secreted. Here we demonstrate that cleavage of this ApoER2 variant does indeed take place, and that the resulting receptor fragment consisting of the entire ligand-binding domain is secreted as soluble polypeptide. This receptor fragment inhibits Reelin signaling in primary neurons, indicating that it can act in a dominant-negative fashion in the regulation of Reelin signaling during embryonic brain development.