Transmembrane Leucine-rich Repeat Protein Research Articles

Trophoblast glycoprotein is required for efficient synaptic vesicle exocytosis from retinal rod bipolar cells.

Rod bipolar cells (RBCs) faithfully transmit light-driven signals from rod photoreceptors in the outer retina to third order neurons in the inner retina. Recently, significant work has focused on the role of leucine-rich repeat (LRR) proteins in synaptic development and signal transduction at RBC synapses. We previously identified trophoblast glycoprotein (TPBG) as a novel transmembrane LRR protein localized to the dendrites and axon terminals of RBCs. We examined the effects on RBC physiology and retinal processing of TPBG genetic knockout in mice using immunofluorescence and electron microscopy, electroretinogram recording, patch-clamp electrophysiology, and time-resolved membrane capacitance measurements. The scotopic electroretinogram showed a modest increase in the b-wave and a marked attenuation in oscillatory potentials in the TPBG knockout. No effect of TPBG knockout was observed on the RBC dendritic morphology, TRPM1 currents, or RBC excitability. Because scotopic oscillatory potentials primarily reflect RBC-driven rhythmic activity of the inner retina, we investigated the contribution of TPBG to downstream transmission from RBCs to third-order neurons. Using electron microscopy, we found shorter synaptic ribbons in TPBG knockout axon terminals in RBCs. Time-resolved capacitance measurements indicated that TPBG knockout reduces synaptic vesicle exocytosis and subsequent GABAergic reciprocal feedback without altering voltage-gated Ca2+ currents. TPBG is required for normal synaptic ribbon development and efficient neurotransmitter release from RBCs to downstream cells. Our results highlight a novel synaptic role for TPBG at RBC ribbon synapses and support further examination into the mechanisms by which TPBG regulates RBC physiology and circuit function.

Biallelic mutations in ELFN1 gene associated with developmental and epileptic encephalopathy and joint laxity

ELFN1, a transmembrane leucine rich repeat protein, is involved in signal transduction in both neural cells and ROD ON-bipolar synaptogenesis. We present three siblings with developmental and epileptic encephalopathy and co-morbidities due to ELFN1 gene mutation; this is the first report in literature defining the human phenotype of ELFN1 gene mutation. Clinical, electrophysiological, and radiological findings along with comprehensive genetic studies of the patients and their family members are presented. Developmental and epileptic encephalopathy, autistic features, pyramidal signs, joint laxity, and dysmorphic features are the characteristic findings of this new clinical entity, involving mainly nervous system and possibly connective tissue. Whole exome sequence analysis followed by Sanger sequencing in all family members revealed disease-causing 8 bp frameshift mutation depicted as NM_001128636.2: c.42_49delGGCCGCCA; p. (Ala15Profs*241) in ELFN1. The variant, located in the signal peptide domain in the ELFN1 gene, was found to be homozygous in three patients, and heterozygous in the parents and three healthy siblings. Segregation analysis in family members together with pathogenicity assessment tools strongly supported the damaging effect of the frameshift variant on the function of the ELFN1 protein. Mutations in ELFN1 gene may be considered in patients with neonatal and infantile-onset epileptic encephalopathy before the full clinical picture is apparent.

Lapsyn controls branch extension and positioning of astrocyte-like glia in the Drosophila optic lobe

Astrocytes have diverse, remarkably complex shapes in different brain regions. Their branches closely associate with neurons. Despite the importance of this heterogeneous glial cell type for brain development and function, the molecular cues controlling astrocyte branch morphogenesis and positioning during neural circuit assembly remain largely unknown. We found that in the Drosophila visual system, astrocyte-like medulla neuropil glia (mng) variants acquire stereotypic morphologies with columnar and layered branching patterns in a stepwise fashion from mid-metamorphosis onwards. Using knockdown and loss-of-function analyses, we uncovered a previously unrecognized role for the transmembrane leucine-rich repeat protein Lapsyn in regulating mng development. lapsyn is expressed in mng and cell-autonomously required for branch extension into the synaptic neuropil and anchoring of cell bodies at the neuropil border. Lapsyn works in concert with the fibroblast growth factor (FGF) pathway to promote branch morphogenesis, while correct positioning is essential for mng survival mediated by gliotrophic FGF signaling.

Abstract 32: The role of Lrig1 signaling in mammary gland development and tumorigenesis

Abstract Lrig1 is a transmembrane leucine-rich repeat protein and a negative regulator of oncogenic receptor tyrosine kinases (RTKs) such as the ErbB family members. The Sweeney lab recently demonstrated that under-expression of Lrig1 predicts poor prognosis in breast cancer. Breast cancer patients with high Lrig1 expression show significantly longer relapse-free survival, identifying Lrig1 as a new prognostic marker. Recently, Lrig1 was found to be a tumor suppressor in intestinal tissue. It was shown that Lrig1 null mice develop ErbB receptor-dependent duodenal adenomas. Based on Lrig1's function as a negative regulator of RTKs and its reported tumor suppressor activity in intestinal epithelium, we hypothesized that Lrig1 null mice would be susceptible to mammary tumorigenesis. To characterize the function of Lrig1 in the mammary gland, we compared mammary development in Lrig1 wild type (+/+) and null (-/-) mice at regular intervals from 4 to 12 weeks. Mice were screened for evidence of hyperplasia, pre-malignant lesions or tumors by whole mount analysis across age and the intensity of specific proteins was surveyed by western blot. Organotypic growth of the mammary epithelial cells from wild type and null mice was examined in three-dimensional cell culture to examine whether any phenotypes are inherent to the epithelium. Since Lrig1 has been defined as a stem cell marker in the intestine, its role in stem cell maintenance was determined by examining the impact of Lrig1 loss on mammary stem cells. Although these studies are ongoing, early results indicate an increase in the amount of Lrig1-target proteins by western blot in the Lrig1-/- mice as opposed to the Lrig1+/+ mice mammary gland tissue. Epithelium from Lrig1-/- mice present an exaggerated branching response when stimulated with TGFα in comparison to Lrig1+/+ epithelium. Our studies also indicate that Lrig1-/- mammary glands are enriched in stem cells when compared to Lrig1+/+ mammary glands. In addition, a number of proliferative lesions were encountered in the mammary gland whole mounts of the Lrig1-/- mice, but not that of the Lrig1+/+ mice, which strongly suggests that Lrig1 plays a growth suppressor role in the mammary gland. Citation Format: Catalina Simion, Qian J. Chen, Charles L. Wilkerson, Hanine Rafidi, Alexander D. Borowsky, Colleen Sweeney. The role of Lrig1 signaling in mammary gland development and tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 32. doi:10.1158/1538-7445.AM2015-32

The unfolded protein response is required for dendrite morphogenesis.

Precise patterning of dendritic fields is essential for the formation and function of neuronal circuits. During development, dendrites acquire their morphology by exuberant branching. How neurons cope with the increased load of protein production required for this rapid growth is poorly understood. Here we show that the physiological unfolded protein response (UPR) is induced in the highly branched Caenorhabditis elegans sensory neuron PVD during dendrite morphogenesis. Perturbation of the IRE1 arm of the UPR pathway causes loss of dendritic branches, a phenotype that can be rescued by overexpression of the ER chaperone HSP-4 (a homolog of mammalian BiP/grp78). Surprisingly, a single transmembrane leucine-rich repeat protein, DMA-1, plays a major role in the induction of the UPR and the dendritic phenotype in the UPR mutants. These findings reveal a significant role for the physiological UPR in the maintenance of ER homeostasis during morphogenesis of large dendritic arbors.

Characterization of microsporidia-induced developmental arrest and a transmembrane leucine-rich repeat protein in Caenorhabditis elegans.

Microsporidia comprise a highly diverged phylum of intracellular, eukaryotic pathogens, with some species able to cause life-threatening illnesses in immunocompromised patients. To better understand microsporidian infection in animals, we study infection of the genetic model organism Caenorhabditis elegans and a species of microsporidia, Nematocida parisii, which infects Caenorhabditis nematodes in the wild. We conducted a targeted RNAi screen for host C. elegans genes important for infection and growth of N. parisii, using nematode larval arrest as an assay for infection. Here, we present the results of this RNAi screen, and our analyses on one of the RNAi hits from the screen that was ultimately not corroborated by loss of function mutants. This hit was an RNAi clone against F56A8.3, a conserved gene that encodes a transmembrane protein containing leucine-rich repeats (LRRs), a domain found in numerous pathogen receptors from other systems. This RNAi clone caused C. elegans to be resistant to infection by N. parisii, leading to reduced larval arrest and lower pathogen load. Characterization of the endogenous F56A8.3 protein revealed that it is expressed in the intestine, localized to the membrane around lysosome-related organelles (LROs), and exists in two different protein isoforms in C. elegans. We used the CRISPR-Cas9 system to edit the F56A8.3 locus and created both a frameshift mutant resulting in a truncated protein and a complete knockout mutant. Neither of these mutants was able to recapitulate the infection phenotypes of the RNAi clone, indicating that the RNAi-mediated phenotypes are due to an off-target effect of the RNAi clone. Nevertheless, this study describes microsporidia-induced developmental arrest in C. elegans, presents results from an RNAi screen for host genes important for microsporidian infection, and characterizes aspects of the conserved F56A8.3 gene and its protein product.

Leucine-rich Repeat and Immunoglobulin Domain-containing Protein-1 (Lrig1) Negative Regulatory Action toward ErbB Receptor Tyrosine Kinases Is Opposed by Leucine-rich Repeat and Immunoglobulin Domain-containing Protein 3 (Lrig3)

Lrig1 is the founding member of the Lrig family of transmembrane leucine-rich repeat proteins, which also includes Lrig2 and Lrig3. Lrig1 is a negative regulator of oncogenic receptor tyrosine kinases, including ErbB and Met receptors, and promotes receptor degradation. Lrig1 has recently emerged as both a tumor suppressor and a key regulator of epidermal and epithelial stem cell quiescence. Despite this, little is known of the mechanisms by which Lrig1 is regulated. Lrig3 was recently reported to increase ErbB receptor expression suggesting that it may function in a manner opposite to Lrig1. In this study, we explore the interaction between Lrig1 and Lrig3 and demonstrate that Lrig1 and Lrig3 functionally oppose one another. Lrig3 opposes Lrig1 negative regulatory activity and stabilizes ErbB receptors. Conversely, Lrig1 destabilizes Lrig3, limiting Lrig3's positive effects on receptors and identifying Lrig3 as a new target of Lrig1. These studies provide new insight into the regulation of Lrig1 and uncover a complex cross-talk between Lrig1 and Lrig3.

The transmembrane LRR protein DMA-1 promotes dendrite branching and growth in C. elegans

Dendrites often adopt complex branched structures. The development and organization of these arbors fundamentally determine the potential input and connectivity of a given neuron. The cell-surface receptors that control dendritic branching remain poorly understood. Here, we show that in Caenorhabditis elegans, a previously uncharacterized transmembrane protein containing extracellular leucine-rich repeat (LRR) domains, which we name DMA-1 (Dendrite-Morphogenesis-Abnormal), promotes dendrite branching and growth. Sustained expression of dma-1 is found only in the elaborately branched sensory neurons PVD and FLP. Genetic analysis showed that loss of dma-1 causes much reduced dendritic arbors while overexpression of dma-1 results in excessive branching. Forced expression of dma-1 in neurons with simple dendrites was sufficient to promote ectopic branching. Animals lacking dma-1 are defective in sensing harsh touch. DMA-1 is the first transmembrane LRR protein to be implicated in dendritic branching and expands the breadth of roles played by LRR receptors in nervous system development.

LRRN6A/LERN1 (leucine-rich repeat neuronal protein 1), a novel gene with enriched expression in limbic system and neocortex.

Human chromosome 15q24-q26 is a very complex genomic region containing several blocks of segmental duplications to which susceptibility to anxiety disorders has been mapped (Gratacos et al., 2001, Cell, 106, 367-379; Pujana et al., 2001, Genome Res., 11, 98-111). Through an in silico gene content analysis of the 15q24-q26 region we have identifie1d a novel gene, LRRN6A (leucine-rich repeat neuronal 6A), and confirmed its location to the centromeric end of this complex region. LRRN6A encodes a transmembrane leucine-rich repeat protein, LERN1 (leucine-rich repeat neuronal protein 1), with similarity to proteins involved in axonal guidance and migration, nervous system development and regeneration processes. The identification of homologous genes to LRRN6A on chromosomes 9 and 19 and the orthologous genes in the mouse genome and other organisms suggests that LERN proteins constitute a novel subfamily of LRR (leucine-rich repeat)-containing proteins. The LRRN6A expression pattern is specific to the central nervous system, highly and broadly expressed during early stages of development and gradually restricted to forebrain structures as development proceeds. Expression level in adulthood is lower in general but remains stable and significantly enriched in the limbic system and cerebral cortex. Taken together, the confirmation of LRRN6A's expression profile, its predicted protein structure and its similarity to nervous system-expressed LRR proteins with essential roles in nervous system development and maintenance suggest that LRRN6A is a novel gene of relevance in the molecular and cellular neurobiology of vertebrates.

Mechanism of inhibition of the Drosophila and mammalian EGF receptors by the transmembrane protein Kekkon 1.

The transmembrane protein Kekkon 1 (Kek1) has previously been shown to act in a negative feedback loop to downregulate the Drosophila Epidermal Growth Factor Receptor (DER) during oogenesis. We show that this protein plays a similar role in other DER-mediated developmental processes. Structure-function analysis reveals that the extracellular Leucine-Rich Repeat (LRR) domains of Kek1 are critical for its function through direct association with DER, whereas its cytoplasmic domain is required for apical subcellular localization. In addition, the use of chimeric proteins between Kek1 extracellular and transmembrane domains fused to DER intracellular domain indicates that Kek1 forms an heterodimer with DER in vivo. To characterize more precisely the mechanism underlying the Kek1/DER interaction, we used mammalian ErbB/EGFR cell-based assays. We show that Kek1 is capable of physically interacting with each of the known members of the mammalian ErbB receptor family and that the Kek1/EGFR interaction inhibits growth factor binding, receptor autophosphorylation and Erk1/2 activation in response to EGF. Finally, in vivo experiments show that Kek1 expression potently suppresses the growth of mouse mammary tumor cells derived from aberrant ErbB receptors activation, but does not interfere with the growth of tumor cells derived from activated Ras. Our results underscore the possibility that Kek1 may be used experimentally to inhibit ErbB receptors and point to the possibility that, as yet uncharacterized, mammalian transmembrane LRR proteins might act as modulators of growth factor signalling.

Synleurin, a novel leucine-rich repeat protein that increases the intensity of pleiotropic cytokine responses

We have identified and characterized a novel single span transmembrane leucine-rich repeat protein, synleurin, that renders cells highly sensitive to the activation by cytokines and lipopolysaccharide (LPS). The major part of the extracellular domain consists of a leucine-rich repeats (LRR) cassette. The LRR central core has 12 analogous LRR repeating modules arranged in a seamless tandem array. The LRRs are most homologous to that of chondroadherin, insulin-like growth factor binding proteins, platelet glycoprotein V, slits, and toll-like receptors. Synleurin expression was detected at low levels in many tissues, including smooth muscle, brain, uterus, pancreas, cartilage, adipose, spleen, and testis. When synleurin is ecotopically expressed in transfected cells, the cells exhibit amplified responses to bFGF, EGF, PDGF-B, IGF-1, IGF-2, and LPS. Synleurin gene ( slrn) maps to human chromosome at 5q12. The name synleurin reflects its synergistic effect on cytokine stimulation and its prominent leucine-rich repeats.

Identification of FLRT1, FLRT2, and FLRT3: A Novel Family of Transmembrane Leucine-Rich Repeat Proteins

FLRT1, FLRT2, and FLRT3 comprise a novel gene family isolated in a screen for extracellular matrix proteins expressed in muscle. The three genes encode putative type I transmembrane proteins, each containing 10 leucine-rich repeats flanked by N-terminal and C-terminal cysteine-rich regions, a fibronectin/collagen-like domain, and an intracellular tail. FLRT1 is expressed in kidney and brain, FLRT2 is expressed in pancreas, skeletal muscle, brain, and heart, and FLRT3 is expressed in kidney, brain, pancreas, skeletal muscle, lung, liver, placenta, and heart. FLRT1 localized to 11q12–q13, FLRT2 to 14q24–q32, and FLRT3 to 20p11. When expressed in SF9 and COS-1 cells, FLRT1 and FLRT2 migrate as 90- and 85-kDa proteins, respectively, and both are glycosylated. Given the overall structure of the three proteins, a function in cell adhesion and/or receptor signaling is predicted.