Membrane structure and internalization dynamics of human Flower isoforms hFWE3 and hFWE4 indicate a conserved endocytic role for hFWE4.

Human Flower (hFWE) isoforms hFWE1-4 are putative transmembrane (TM) proteins that reportedly mediate fitness comparisons during cell competition through extracellular display of their C-terminal tails. Isoform topology, subcellular localization, and duration of plasma membrane presentation are essential to this function. However, disagreement persists regarding the structure of orthologous fly and mouse FWEs, and experimental evidence for hFWE isoform subcellular localization or membrane structure is lacking. Here, we used AlphaFold2 and subsequent molecular dynamics-based structural predictions to construct epitope-tagged hFWE3 and hFWE4, the most abundant human isoforms, for experimental determination of their structure and internalization dynamics. We demonstrate that hFWE3 resides in the membrane of the endoplasmic reticulum (ER), while hFWE4 partially colocalizes with Rab4-, Rab5-, and Rab11-positive vesicles as well as with the plasma membrane. An array of imaging techniques revealed that hFWE4 positions both N- and C-terminal tails and a loop between second and third TM segments within the cytosol, while small (4-12aa) loops between the first and second and the third and fourth TM segments are either exposed to the extracellular space or within the lumen of cytoplasmic vesicles. Similarly, we found hFWE3 positions both N- and C-terminal tails in the cytosol, while a short loop between TM domains extends into the ER lumen. Finally, we demonstrate that hFWE4 exists only transiently at the cell surface and is rapidly internalized in an AP-2- and dynamin-1-dependent manner. Collectively, these data are consistent with a conserved role for hFWE4 in endocytic processes.

A LGR5 reporter pig model closely resembles human intestine for improved study of stem cells in disease.

Intestinal epithelial stem cells (ISCs) are responsible for intestinal epithelial barrier renewal; thereby, ISCs play a critical role in intestinal pathophysiology research. While transgenic ISC reporter mice are available, advanced translational studies lack a large animal model. This study validates ISC isolation in a new porcine Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5) reporter line and demonstrates the use of these pigs as a novel colorectal cancer (CRC) model. We applied histology, immunofluorescence, fluorescence-activated cell sorting, flow cytometry, gene expression quantification, and 3D organoid cultures to whole tissue and single cells from the duodenum, jejunum, ileum, and colon of LGR5-H2B-GFP and wild-type pigs. Ileum and colon LGR5-H2B-GFP, healthy human, and murine biopsies were compared by mRNA fluorescent in situ hybridization (FISH). To model CRC, adenomatous polyposis coli (APC) mutation was induced by CRISPR/Cas9 editing in porcine LGR5-H2B-GFP colonoids. Crypt-base, green fluorescent protein (GFP) expressing cells co-localized with ISC biomarkers. LGR5-H2B-GFPhi cells had significantly higher LGR5 expression (p < .01) and enteroid forming efficiency (p < .0001) compared with LGR5-H2B-GFPmed/lo/neg cells. Using FISH, similar LGR5, OLFM4, HOPX, LYZ, and SOX9 expression was identified between human and LGR5-H2B-GFP pig crypt-base cells. LGR5-H2B-GFP/APCnull colonoids had cystic growth in WNT/R-spondin-depleted media and significantly upregulated WNT/ß-catenin target gene expression (p < .05). LGR5+ ISCs are reproducibly isolated in LGR5-H2B-GFP pigs and used to model CRC in an organoid platform. The known anatomical and physiologic similarities between pig and human, and those shown by crypt-base FISH, underscore the significance of this novel LGR5-H2B-GFP pig to translational ISC research.

The use of histotripsy as intratumoral immunotherapy beyond tissue ablation-the rationale for exploring the immune effects of histotripsy.

Mechanical high-intensity focused ultrasound (M-HIFU), which includes histotripsy, is a non-ionizing, non-thermal ablation technology that can be delivered by noninvasive methods. Because acoustic cavitation is the primary mechanism of tissue disruption, histotripsy is distinct from the conventional HIFU techniques resulting in hyperthermia and thermal injury. Phase I human trials have shown the initial safety and efficacy of histotripsy in treating patients with malignant liver tumors. In addition to tissue ablation, a promising benefit of M-HIFU has been stimulating a local and systemic antitumor immune response in preclinical models and potentially in the Phase I trial. Preclinical studies combining systemic immune therapies appear promising, but clinical studies of combinations have been complicated by systemic toxicities. Consequently, combining M-HIFU with systemic immunotherapy has been demonstrated in preclinical models and may be testing in future clinical studies. An additional alternative is to combine intratumoral M-HIFU and immunotherapy using microcatheter-placed devices to deliver both M-HIFU and immunotherapy intratumorally. The promise of M-HIFU as a component of anti-cancer therapy is promising, but as forms of HIFU are tested in preclinical and clinical studies, investigators should report not only the parameters of the energy delivered but also details of the preclinical models to enable analysis of the immune responses. Ultimately, as clinical trials continue, clinical responses and immune analysis of patients undergoing M-HIFU including forms of histotripsy will provide opportunities to optimize clinical responses and to optimize application and scheduling of M-HIFU in the context of the multi-modality care of the cancer patient.

Author Correction: Eliminating dissolution of platinum-based electrocatalysts at the atomic scale.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Eliminating dissolution of platinum-based electrocatalysts at the atomic scale.

A remaining challenge for the deployment of proton-exchange membrane fuel cells is the limited durability of platinum (Pt) nanoscale materials that operate at high voltages during the cathodic oxygen reduction reaction. In this work, atomic-scale insight into well-defined single-crystalline, thin-film and nanoscale surfaces exposed Pt dissolution trends that governed the design and synthesis of durable materials. A newly defined metric, intrinsic dissolution, is essential to understanding the correlation between the measured Pt loss, surface structure, size and ratio of Pt nanoparticles in a carbon (C) support. It was found that the utilization of a gold (Au) underlayer promotes ordering of Pt surface atoms towards a (111) structure, whereas Au on the surface selectively protects low-coordinated Pt sites. This mitigation strategy was applied towards 3 nm Pt3Au/C nanoparticles and resulted in the elimination of Pt dissolution in the liquid electrolyte, which included a 30-fold durability improvement versus 3 nm Pt/C over an extended potential range up to 1.2 V.

Fringe visibility errors resulting from dual object imaging for stellar interferometers.

Practical stellar interferometry for space domain awareness is challenged by the relative motions of orbital objects and telescope arrays that require array phasing using guide stars. An orbital object's image sensitivity to the location and brightness of the guide star is problematic, possibly resulting in a degraded resolution or loss of image content when both objects fall within the interferometer's field of view. We characterized an orbital object's visibility using visibility contrast to noise ratios (CNRΔv) as a performance metric for orbital object image quality. Experimental validations included orbital object visibility measurements for dual binary pinholes that were scaled in size and brightness individually to match expected interferometer data collection scenarios. We show agreement in CNRΔv results, indicating resolvable orbital object signals during periods of collection when signal contributions from both the orbital object and guide star are present. Expanding presented results to imaging interferometers, we discuss how dual object imaging could degrade performance under the scenarios examined.

Distinct cortical and striatal actions of a ß-arrestin-biased dopamine D2 receptor ligand reveal unique antipsychotic-like properties.

The current dopamine (DA) hypothesis of schizophrenia postulates striatal hyperdopaminergia and cortical hypodopaminergia. Although partial agonists at DA D2 receptors (D2Rs), like aripiprazole, were developed to simultaneously target both phenomena, they do not effectively improve cortical dysfunction. In this study, we investigate the potential for newly developed ß-arrestin2 (ßarr2)-biased D2R partial agonists to simultaneously target hyper- and hypodopaminergia. Using neuron-specific ßarr2-KO mice, we show that the antipsychotic-like effects of a ßarr2-biased D2R ligand are driven through both striatal antagonism and cortical agonism of D2R-ßarr2 signaling. Furthermore, ßarr2-biased D2R agonism enhances firing of cortical fast-spiking interneurons. This enhanced cortical agonism of the biased ligand can be attributed to a lack of G-protein signaling and elevated expression of ßarr2 and G protein-coupled receptor (GPCR) kinase 2 in the cortex versus the striatum. Therefore, we propose that ßarr2-biased D2R ligands that exert region-selective actions could provide a path to develop more effective antipsychotic therapies.

Inhibiting clathrin-mediated endocytosis of the leucine-rich G protein-coupled receptor-5 diminishes cell fitness.

The leucine-rich G protein-coupled receptor-5 (LGR5) is expressed in adult tissue stem cells of many epithelia, and its overexpression is negatively correlated with cancer prognosis. LGR5 potentiates WNT/ß-catenin signaling through its unique constitutive internalization property that clears negative regulators of the WNT-receptor complex from the membrane. However, both the mechanism and physiological relevance of LGR5 internalization are unclear. Therefore, a natural product library was screened to discover LGR5 internalization inhibitors and gain mechanistic insight into LGR5 internalization. The plant lignan justicidin B blocked the constitutive internalization of LGR5. Justicidin B is structurally similar to more potent vacuolar-type H+-ATPase inhibitors, which all inhibited LGR5 internalization by blocking clathrin-mediated endocytosis. We then tested the physiological relevance of LGR5 internalization blockade in vivo A LGR5-rainbow (LBOW) mouse line was engineered to express three different LGR5 isoforms along with unique fluorescent protein lineage reporters in the same mouse. In this manner, the effects of each isoform on cell fate can be simultaneously assessed through simple fluorescent imaging for each lineage reporter. LBOW mice express three different forms of LGR5, a wild-type form that constitutively internalizes and two mutant forms whose internalization properties have been compromised by genetic perturbations within the carboxyl-terminal tail. LBOW was activated in the intestinal epithelium, and a year-long lineage-tracing course revealed that genetic blockade of LGR5 internalization diminished cell fitness. Together these data provide proof-of-concept genetic evidence that blocking the clathrin-mediated endocytosis of LGR5 could be used to pharmacologically control cell behavior.

Lgr4 and Lgr5 drive the formation of long actin-rich cytoneme-like membrane protrusions.

Embryonic development and adult tissue homeostasis require precise information exchange between cells and their microenvironment to coordinate cell behavior. A specialized class of ultra-long actin-rich filopodia, termed cytonemes, provides one mechanism for this spatiotemporal regulation of extracellular cues. We provide here a mechanism whereby the stem-cell marker Lgr5, and its family member Lgr4, promote the formation of cytonemes. Lgr4- and Lgr5-induced cytonemes exceed lengths of 80 µm, are generated through stabilization of nascent filopodia from an underlying lamellipodial-like network and functionally provide a pipeline for the transit of signaling effectors. As proof-of-principle, we demonstrate that Lgr5-induced cytonemes act as conduits for cell signaling by demonstrating that the actin motor and filopodial cargo carrier protein myosin X (Myo10) and the G-protein-coupled receptor (GPCR) signaling effector ß-arrestin-2 (Arrb2) transit into cytonemes. This work delineates a biological function for Lgr4 and Lgr5 and provides the rationale to fully investigate Lgr4 and Lgr5 function and cytonemes in mammalian stem cell and cancer stem cell behavior.

BN Doping and the Photochemistry of Polyaromatic Hydrocarbons: Photocyclization of Hexaphenyl Benzene and Hexaphenyl Borazine.

Boron-nitrogen doping of polyaromatic hydrocarbon (PAH) materials can be used to tune their electronic properties while preserving the structural characteristics of pure hydrocarbons. Many multicycle PAHs can be synthesized photochemically; in contrast, very little is known about the photochemistry of their BN-doped counterparts. We present results of fs, ns, and µs time-resolved spectroscopic studies on the photoinduced dynamics of hexaphenyl benzene and hexaphenyl borazine in order to examine how BN doping alters photochemical C-C bond formation via 6π electrocyclization as well as the stability of resulting cyclized structures. Ultrafast measurements reveal photoinduced behaviors reflecting differences in excited-state decay pathways for the two molecules, with hexaphenyl borazine relaxing from its excited state with a rate that is 2 orders of magnitude faster than that of hexaphenyl benzene (3.0 vs 428 ps). Tetraphenyl dihydrotriphenylene generated from hexaphenyl benzene is observed to reopen with a ∼2 µs lifetime controlled by entropic stabilization of the cyclized structure; in contrast, photoinduced dynamics appear to be complete within 100 ps after excitation of hexaphenyl borazine. This significant difference in photochemical dynamics is reflected in the cyclodehydrogentation yields obtained for the two reactants (25 vs 0% for hexaphenyl benzene and borazine, respectively). Quantum-chemical computations predict that BN doping gives rise to energetic destabilization and increased singlet diradical character in cyclized structures. These findings indicate that the polarized BN bonds of the borazine core adversely impact photochemical bond formation relative to analogous hydrocarbons.

Excited-State Deactivation Pathways and the Photocyclization of BN-Doped Polyaromatics.

Boron-nitrogen doping of polyaromatic hydrocarbons (PAH), such as borazine-core hexabenzocoronene, presents possibilities for tuning the properties of organic electronics and nanographene materials while preserving structural characteristics of pure hydrocarbons. Previous photochemical studies have demonstrated extension of a borazine-core PAH network (1,2:3,4:5,6-tris(o,o'-biphenylylene)borazine, 1) by photoinduced cyclodehydrogenation. We present steady-state and femtosecond-to-microsecond resolved spectroscopic studies of the photophysics of 1 and a related borazine-core PAH in order to characterize competing excited-state relaxation pathways that determine the efficacy of bond formation by photocyclization. Transient spectra evolve on time scales consistent with S1 fluorescence lifetimes (1-3 ns) to features that persist onto microsecond time scales. Nanosecond-resolved oxygen-quenching measurements reveal that long-lived metastable states are triplets rather than cyclized products. Determination of fluorescence and triplet quantum yields reveal that photochemical bond formation is a minor channel in the relaxation of 1 (∼5% or less), whereas highly efficient fluorescence and intersystem crossing result in negligible photoinduced bond formation in more extended borazine-core networks. Results of computational investigations at the RICC2 level reveal sizable barriers to cyclization on the S1 potential energy surfaces consistent with quantum yields deduced from experiment. Together these barriers and competing photophysical pathways limit the efficiency of photochemical synthesis of BN-doped polyaromatics.

Aminofluorination of Cyclopropanes: A Multifold Approach through a Common, Catalytically Generated Intermediate.

We have discovered a highly regioselective aminofluorination of cyclopropanes. Remarkably, four unique sets of conditions-two photochemical, two purely chemical-generated the same aminofluorinated adducts in good to excellent yields. The multiple, diverse ways in which the reaction could be initiated provided valuable clues that led to the proposal of a "unifying" chain propagation mechanism beyond initiation, tied by a common intermediate. In all, the proposed mechanism herein is substantiated by product distribution studies, kinetic analyses, LFERs, Rehm-Weller estimations of ΔGET, competition experiments, KIEs, fluorescence data, and DFT calculations. From a more physical standpoint, transient-absorption experiments have allowed direct spectroscopic observation of radical ion intermediates (previously only postulated or probed indirectly in photochemical fluorination systems) and, consequently, have provided kinetic support for chain propagation. Lastly, calculations suggest that solvent may play an important role in the cyclopropane ring-opening step.

Structural Control of Nonadiabatic Photochemical Bond Formation: Photocyclization in Structurally Modified ortho-Terphenyls.

Understanding how molecular structure impacts the shapes of potential energy surfaces and prospects for nonadiabatic photochemical dynamics is critical for predicting and controlling the chemistry of molecular excited states. Ultrafast transient absorption spectroscopy was used to interrogate photoinduced, nonadiabatic 6π cyclization of a collection of ortho-terphenyls (OTP) modified with alkyl substituents of different sizes and electron-donating/withdrawing character positioned on its central and pendant phenyl rings. OTP alkylated at the 4,4″ and 4',5' positions of the pendant and central rings, respectively, exhibiting biphasic excited-state relaxation; this is qualitatively similar to relaxation of OTP itself, including a fast decrease in excited-state absorption (τ1 = 1-4 ps) followed by formation of metastable cyclized photoproducts (τ2 = 3-47 ps) that share common characteristic spectroscopic features for all substitutions despite variations in chemical nature of the substituents. By contrast, anomalous excited-state dynamics are observed for 3',6'dimethyl-OTP, in which the methyl substituents crowd the pendant rings sterically; time-resolved spectral dynamics and low photochemical reactivity with iodine reveal that methylation proximal to the pendant rings impedes nonadiabatic cyclization. Results from transient measurements and quantum-chemical calculations are used to decipher the nature of excited state relaxation mechanisms in these systems and how they are perturbed by mechanical, electronic, and steric interactions induced by substituents.

A rapid and affordable screening platform for membrane protein trafficking.

BACKGROUND: Membrane proteins regulate a diversity of physiological processes and are the most successful class of targets in drug discovery. However, the number of targets adequately explored in chemical space and the limited resources available for screening are significant problems shared by drug-discovery centers and small laboratories. Therefore, a low-cost and universally applicable screen for membrane protein trafficking was developed. RESULTS: This high-throughput screen (HTS), termed IRFAP-HTS, utilizes the recently described MarsCy1-fluorogen activating protein and the near-infrared and membrane impermeant fluorogen SCi1. The cell surface expression of MarsCy1 epitope-tagged receptors can be visualized by simple addition of SCi1. User-friendly, rapid, and quantitative detection occurs on a standard infrared western-blotting scanner. The reliability and robustness of IRFAP-HTS was validated by confirming human vasopressin-2 receptor and dopamine receptor-2 trafficking in response to agonist or antagonist. The IRFAP-HTS screen was deployed against the leucine-rich G protein-coupled receptor-5 (Lgr5). Lgr5 is expressed in stem cells, modulates Wnt/ß-catenin signaling, and is therefore a promising drug target. However, small molecule modulators have yet to be reported. The constitutive internalization of Lgr5 appears to be one primary mode through which its function is regulated. Therefore, IRFAP-HTS was utilized to screen 11,258 FDA-approved and drug-like small molecules for those that antagonize Lgr5 internalization. Glucocorticoids were found to potently increase Lgr5 expression at the plasma membrane. CONCLUSION: The IRFAP-HTS platform provides a versatile solution for screening more targets with fewer resources. Using only a standard western-blotting scanner, we were able to screen 5,000 compounds per hour in a robust and quantitative assay. Multi-purposing standardly available laboratory equipment eliminates the need for idiosyncratic and more expensive high-content imaging systems. The modular and user-friendly IRFAP-HTS is a significant departure from current screening platforms. Small laboratories will have unprecedented access to a robust and reliable screening platform and will no longer be limited by the esoteric nature of assay development, data acquisition, and post-screening analysis. The discovery of glucocorticoids as modulators for Lgr5 trafficking confirms that IRFAP-HTS can accelerate drug-discovery and drug-repurposing for even the most obscure targets.

Structural control of nonadiabatic bond formation: the photochemical formation and stability of substituted 4a,4b-dihydrotriphenylenes.

Nonadiabatic photocyclization makes bonds and is the first step in the photoinduced cyclodehydrogenation of ortho-arenes to yield polycyclic aromatic hydrocarbons. How molecular structure alters potential-energy landscapes, excited-state dynamics, and stabilities of reactants and intermediates underlies the feasibility of desirable photochemistry. In order to gain insight into these structure-dynamics relationships, we have used femtosecond transient absorption spectroscopy (TAS) to examine photoinduced dynamics of 1,2,3-triphenylbenzene (TPB) and ortho-quaterphenyl (OQTP), phenyl-subsituted analogues of ortho-terphenyl (OTP). Dynamics of TPB and OTP are quite similar: TPB exhibits fast (7.4 ps) excited-state decay with concomitant formation and vibrational relaxation of 9-phenyl-dihydrotriphenylene (9-phenyl DHT). In contrast, photoexcited OQTP exhibits multistate kinetics leading to formation of 1-phenyl DHT. Excited-state calculations reveal the existence of two distinct minima on the OQTP S1 surface and, together with photophysical data, support a mechanism involving both direct cyclization by way of an asymmetric structure and indirect cyclization by way of a symmetric quinoid-like minimum. Temperature-dependent nanosecond TAS was utilized to assess the relative stabilities of intermediates, substantiating the observed trend in photochemical reactivity OTP > OQTP > TPB. In total, this work demonstrates how specific structural variations alter the course of the excited-state dynamics and photoproduct stability that underlies desired photochemistry.

Deletion of GSK3ß in D2R-expressing neurons reveals distinct roles for ß-arrestin signaling in antipsychotic and lithium action.

Several studies in rodent models have shown that glycogen synthase kinase 3 ß (GSK3ß) plays an important role in the actions of antispychotics and mood stabilizers. Recently it was demonstrated that GSK3ß through a ß-arrestin2/protein kinase B (PKB or Akt)/protein phosphatase 2A (PP2A) signaling complex regulates dopamine (DA)- and lithium-sensitive behaviors and is required to mediate endophenotypes of mania and depression in rodents. We have previously shown that atypical antipsychotics antagonize DA D2 receptor (D2R)/ß-arrestin2 interactions more efficaciously than G-protein-dependent signaling, whereas typical antipsychotics inhibit both pathways with similar efficacy. To elucidate the site of action of GSK3ß in regulating DA- or lithium-sensitive behaviors, we generated conditional knockouts of GSK3ß, where GSK3ß was deleted in either DA D1- or D2-receptor-expressing neurons. We analyzed these mice for behaviors commonly used to test antipsychotic efficacy or behaviors that are sensitive to lithium treatment. Mice with deletion of GSK3ß in D2 (D2GSK3ß(-/-)) but not D1 (D1GSK3ß(-/-)) neurons mimic antipsychotic action. However, haloperidol (HAL)-induced catalepsy was unchanged in either D2GSK3ß(-/-) or D1GSK3ß(-/-) mice compared with control mice. Interestingly, genetic stabilization of ß-catenin, a downstream target of GSK3ß, in D2 neurons did not affect any of the behaviors tested. Moreover, D2GSK3ß(-/-) or D1GSK3ß(-/-) mice showed similar responses to controls in the tail suspension test (TST) and dark-light emergence test, behaviors which were previously shown to be ß-arrestin2- and GSK3ß-dependent and sensitive to lithium treatment. Taken together these studies suggest that selective deletion of GSK3ß but not stabilization of ß-catenin in D2 neurons mimics antipsychotic action without affecting signaling pathways involved in catalepsy or certain mood-related behaviors.

Multimetallic core/interlayer/shell nanostructures as advanced electrocatalysts.

The fine balance between activity and durability is crucial for the development of high performance electrocatalysts. The importance of atomic structure and compositional gradients is a guiding principle in exploiting the knowledge from well-defined materials in the design of novel class of core-shell electrocatalysts comprising Ni core, Au interlayer, and PtNi shell (Ni@Au@PtNi). This multimetallic system is found to have the optimal balance of activity and durability due to the synergy between the stabilizing effect of subsurface Au and modified electronic structure of surface Pt through interaction with subsurface Ni atoms. The electrocatalysts with Ni@Au@PtNi core-interlayer-shell structure exhibit high intrinsic and mass activities as well as superior durability for the oxygen reduction reaction with less than 10% activity loss after 10,000 potential cycles between 0.6 and 1.1 V vs the reversible hydrogen electrode.

Dealloying of noble-metal alloy nanoparticles.

Dealloying is currently used to tailor the morphology and composition of nanoparticles and bulk solids for a variety of applications including catalysis, energy storage, sensing, actuation, supercapacitors, and radiation damage resistant materials. The known morphologies, which evolve on dealloying of nanoparticles, include core-shell, hollow core-shell, and porous nanoparticles. Here we present results examining the fixed voltage dealloying of AgAu alloy particles in the size range of 2-6 and 20-55 nm. High-angle annular dark-field scanning transmission electron microcopy, energy dispersive, and electron energy loss spectroscopy are used to characterize the size, morphology, and composition of the dealloyed nanoparticles. Our results demonstrate that above the potential corresponding to Ag(+)/Ag equilibrium only core-shell structures evolve in the 2-6 nm diameter particles. Dealloying of the 20-55 nm particles results and in the formation of porous structures analogous to the behavior observed for the corresponding bulk alloy. A statistical analysis that includes the composition and particle size distributions characterizing the larger particles demonstrates that the formation of porous nanoparticles occurs at a well-defined thermodynamic critical potential.

Constitutive internalization of the leucine-rich G protein-coupled receptor-5 (LGR5) to the trans-Golgi network.

LGR5 is a Wnt pathway associated G protein-coupled receptor (GPCR) that serves as a molecular determinant of stem cells in numerous tissues including the intestine, stomach, hair follicle, eye, and mammary gland. Despite its importance as a marker for this critical niche, little is known about LGR5 signaling nor the biochemical mechanisms and receptor determinants that regulate LGR5 membrane expression and intracellular trafficking. Most importantly, in cells LGR5 is predominantly intracellular, yet the mechanisms underlying this behavior have not been determined. In this work we elucidate a precise trafficking program for LGR5 and identify the motif at its C terminus that is responsible for the observed constitutive internalization. We show that this process is dependent upon dynamin GTPase activity and find that wild-type full-length LGR5 rapidly internalizes into EEA1- and Rab5-positive endosomes. However, LGR5 fails to rapidly recycle to the plasmid membrane through Rab4-positive vesicles, as is common for other GPCRs. Rather, internalized LGR5 transits through Rab7- and Rab9-positive vesicles, co-localizes in vesicles with Vps26, a retromer complex component that regulates retrograde trafficking to the trans-Golgi network (TGN) and reaches a steady-state distribution in the TGN within 2 h. Using mutagenesis, particularly of putative phosphorylation sites, we show that the amino acid pair, serine 861 and 864, is the principal C-tail determinant that mediates LGR5 constitutive internalization. The constitutive internalization of LGR5 to the TGN suggests the existence of novel biochemical roles for its Wnt pathway related, but ill defined signaling program.