Basolateral targeting by leucine-rich repeat domains in epithelial cells

The asymmetric distribution of proteins to basolateral and apical membranes is an important feature of epithelial cell polarity. To investigate how basolateral LAP proteins (LRR (for leucine-rich repeats) and PDZ (for PSD-95/Discs-large/ZO-1), which play key roles in cell polarity, reach their target membrane, we carried out a structure–function study of three LAP proteins: Caenorhabditis elegans LET-413, human Erbin and human Scribble (hScrib). Deletion and point mutation analyses establish that their LRR domain is crucial for basolateral membrane targeting. This property is specific to the LRR domain of LAP proteins, as the non-LAP protein SUR-8 does not localize at the basolateral membrane of epithelial cells, despite having a closely related LRR domain. Importantly, functional studies of LET-413 in C. elegans show that although its PDZ domain is dispensable during embryogenesis, its LRR domain is essential. Our data establish a novel paradigm for protein localization by showing that a subset of LRR domains direct subcellular localization in polarized cells.     

Leucine-rich repeat region of decorin binds to filamin-A

Decorin is a member of the family of small leucine-rich proteoglycans found in the extracellular matrix and has an important role in promoting fiber formation and in controlling cell proliferation. Here, we have investigated whether the leucine-rich repeat (LRR) region of decorin interacts with proteins from human lung fibroblasts by using a yeast two-hybrid assay. We report that the LRR region of decorin interacts with the cytoskeletal protein, filamin-A (ABP-280), a peripheral cytoplasmic protein. This interaction is dependent on the 288 carboxyl-terminal amino acids of filamin-A, which correspond to repeats 22-24 of its conserved beta-sheet structure. We also show that the recombinant LRR region of decorin binds to filamin-A in vitro, and that the deglycosylated core protein of decorin coprecipitates with filamin-A, whereas intact decorin does not. Together, these results suggest that proteins containing the LRR motif that interact with filamin-A may be present in the cytoplasm or at the plasma membrane.     

Factor XI interacts with the leucine-rich repeats of glycoprotein Ibalpha on the activated platelet

Factor XI (FXI) binds specifically and reversibly to high affinity sites on the surface of stimulated platelets (Kd app of approximately 10 nm; Bmax of approximately 1,500 sites/platelet) utilizing residues exposed on the Apple 3 domain in the presence of high molecular weight kininogen and Zn2+ or prothrombin and Ca2+. Because the FXI receptor in the platelet membrane is contained within the glycoprotein Ibalpha subunit of the glycoprotein Ib-IX-V complex (Baglia, F. A., Badellino, K. O., Li, C. Q., Lopez, J. A., and Walsh, P. N. (2002) J. Biol. Chem. 277, 1662-1668), we utilized mocarhagin, a cobra venom metalloproteinase, to generate a fragment (His1-Glu282) of glycoprotein Ibalpha that contains the leucine-rich repeats of the NH2-terminal globular domain and excludes the macroglycopeptide portion of glycocalicin, the soluble extracytoplasmic portion of glycoprotein Ibalpha. This fragment was able to compete with FXI for binding to activated platelets (Ki of 3.125 +/- 0.25 nm) with a potency similar to that of intact glycocalicin (Ki of 3.72 +/- 0.30 nm). However, a synthetic glycoprotein Ibalpha peptide, Asp269-Asp287, containing a thrombin binding site had no effect on the binding of FXI to activated platelets. Moreover, the binding of 125I-labeled thrombin to glycocalicin was unaffected by the presence of FXI at concentrations up to 10(-5) m. The von Willebrand factor A1 domain, which binds the leucine-rich repeats, inhibited the binding of FXI to activated platelets. Thus, we examined the effect of synthetic peptides of each of the seven leucine-rich repeats on the binding of 125I-FXI to activated platelets. All leucine-rich repeat (LRR) peptides derived from glycoprotein Ibalpha were able to inhibit FXI binding to activated platelets in the following order of decreasing potency: LRR7, LRR1, LRR4, LRR5, LRR6, LRR3, and LRR2. However, the leucine-rich repeat synthetic peptides derived from glycoprotein Ibbeta and Toll protein had no effect. We conclude that FXI binds to glycoprotein Ibalpha at sites comprising the leucine-rich repeat sequences within the NH2-terminal globular domain that are separate and distinct from the thrombin-binding site.     

Requirement of the cytosolic interaction between PATHOGENESIS-RELATED PROTEIN10 and LEUCINE-RICH REPEAT PROTEIN1 for cell death and defense signaling in pepper

Plants recruit innate immune receptors such as leucine-rich repeat (LRR) proteins to recognize pathogen attack and activate defense genes. Here, we identified the pepper (Capsicum annuum) pathogenesis-related protein10 (PR10) as a leucine-rich repeat protein1 (LRR1)-interacting partner. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirmed the specific interaction between LRR1 and PR10 in planta. Avirulent Xanthomonas campestris pv vesicatoria infection induces PR10 expression associated with the hypersensitive cell death response. Transient expression of PR10 triggers hypersensitive cell death in pepper and Nicotiana benthamiana leaves, which is amplified by LRR1 coexpression as a positive regulator. LRR1 promotes the ribonuclease activity and phosphorylation of PR10, leading to enhanced cell death signaling. The LRR1-PR10 complex is formed in the cytoplasm, resulting in its secretion into the apoplastic space. Engineered nuclear confinement of both proteins revealed that the cytoplasmic localization of the PR10-LRR1 complex is essential for cell death-mediated defense signaling. PR10/LRR1 silencing in pepper compromises resistance to avirulent X. campestris pv vesicatoria infection. By contrast, PR10/LRR1 overexpression in Arabidopsis thaliana confers enhanced resistance to Pseudomonas syringae pv tomato and Hyaloperonospora arabidopsidis. Together, these results suggest that the cytosolic LRR-PR10 complex is responsible for cell death-mediated defense signaling.     

OMgp LRR281～228：OMgp神经突起生长抑制功能的主要结构域

OMgp(oligodendrocyte-myelin glycoprotein)可以通过与MAG、nogo-66等神经再生抑制因子竞争结合同一受体NgR而诱使生长锥溃变和抑制神经突起的生长。以前的研究表明,在OMgp与NgR结合抑制神经突起生长的过程中,OMgp的亮氨酸富含重复序列(LRR)是必需的。为进一步了解OMgp LRR在神经突起生长中的作用及其结构与功能之间的关系,采用PCR-定点突变法对OMgp LRR结构域分段删除,表达了删除不同基因片段后的OMgp LRR蛋白,通过对表达有NgR的CHO细胞(NgR-CHO)的黏附实验和对原代培养神经细胞的抑制实验对其功能进行了研究。结果显示,分别删除了OMgp 25～56、57～133、134～180位氨基酸的OMgp LRR蛋白仍具有结合NgR-CHO和抑制原代培养的神经元突起生长的作用;而删除了第181～228位氨基酸的OMgp LRR蛋白则失去了对原代培养神经元的生长抑制作用,但仍然具有结合NgR的能力。表明OMgp181～228在OMgp的功能中具有重要的意义。删除了第181～228位氨基酸的OMgp LRR蛋白可望作为OMgp的竞争性抑制剂,用于中枢神经系统损伤后神经再生的治疗。     

Peptides derived from human decorin leucine-rich repeat 5 inhibit angiogenesis

Excessive angiogenesis is involved in many human diseases, and inhibiting angiogenesis is an important area of drug development. There have been conflicting reports as to whether decorin could function as an angiogenic inhibitor when used as an extracellular soluble factor. In this study, we demonstrated that not only purified decorin but also the 26-residue leucine-rich repeat 5 (LRR5) of decorin core protein functions as angiogenesis inhibitor by inhibiting both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor-induced angiogenesis. Peptide LRR5 inhibited angiogenesis through multiple mechanisms, including inhibiting VEGF-stimulated endothelial cell (EC) migration, tube formation on Matrigel, cell attachment to fibronectin, as well as induction of EC apoptosis without significantly affecting their proliferation. We further demonstrated that different subregions of LRR5 inhibited different aspects of angiogenesis, with the middle region (LRR5M, 12 residues) inhibiting endothelial cell tube formation up to 1000 times more potently than LRR5. Although the C-terminal region (LRR5C) potently inhibited VEGF-stimulated endothelial cell migration, the N-terminal region (LRR5N) is as active as LRR5 in inhibiting endothelial cell attachment to fibronectin. Although both LRR5M and LRR5N induced EC apoptosis dose-dependently similar to LRR5 through a caspase-dependent pathway, LRR5C has no such function. We further showed that the inhibition of tube formation by LRR5 and LRR5M is linked with their ability to suppress VEGF-induced focal adhesion kinase phosphorylation and the assembly of focal adhesions and actin stress fibers in ECs, but not their ability to interfere with endothelial cell attachment to the matrix. Circular dichroism studies revealed that LRR5 undergoes an inter-conversion between 3(10) helix and beta-sheet structure in solution, a characteristic potentially important for its anti-angiogenic activity. Peptide LRR5 and its derivatives are therefore novel angiogenesis inhibitors that may serve as prototypes for further development into anti-angiogenic drugs.     

Leucine-rich repeat (LRR) proteins: integrators of pattern recognition and signaling in immunity

The leucine-rich repeats (LRR)-containing domain is evolutionarily conserved in many proteins associated with innate immunity in plants, invertebrates and vertebrates. Serving as a first line of defense, the innate immune response is initiated through the sensing of pathogen-associated molecular patterns (PAMPs). In plants, NBS (nucleotide-binding site)-LRR proteins provide recognition of pathogen products of avirulence (AVR) genes. LRRs also promote interaction between LRR proteins as observed in receptor-coreceptor complexes. In mammals, toll-like receptors (TLRs) and NOD-like receptors (NLRs) through their LRR domain, sense molecular determinants from a structurally diverse set of bacterial, fungal, parasite and viral-derived components. In humans, at least 34 LRR proteins are implicated in diseases. Most LRR domains consist of 2-45 leucine-rich repeats, with each repeat about 20-30 residues long. Structurally, LRR domains adopt an arc or horseshoe shape, with the concave face consisting of parallel β-strands and the convex face representing a more variable region of secondary structures including helices. Apart from the TLRs and NLRs, most of the 375 human LRR proteins remain uncharacterized functionally. We incorporated computational and functional analyses to facilitate multifaceted insights into human LRR proteins and outline a few approaches here.     

Fine tuning cellular recognition

The formation of complex tissues during embryonic development is often accompanied by directed cellular migration towards a target tissue. Specific mutual recognition between the migrating cell and its target tissue leads to the arrest of the cell migratory behavior and subsequent contact formation between the two interacting cell types. Recent studies implicated a novel family of surface proteins containing a trans-membrane domain and single leucine-rich repeat (LRR) domain in inter-cellular recognition and the arrest of cell migration. Here, I describe the involvement of a novel LRR surface protein, LRT, in targeting migrating muscles towards their corresponding tendon cells in the Drosophila embryo. LRT is specifically expressed by the target tendon cells, and is essential for arresting the migratory behavior of the muscle cells. Additional studies in Drosophila S2 cultured cells suggest that LRT forms a protein complex with the Roundabout (Robo) receptor, essential for guiding muscles towards their tendon partners. Genetic analysis supports a model in which LRT performs its activity non-autonomously through its interaction with the Robo receptors expressed on the muscle surfaces. These results suggest a novel mechanism of intercellular recognition through interactions between LRR family members and Robo receptors.     

Reciprocal cross-talk between Nod2 and TAK1 signaling pathways

Mutations in the leucine-rich repeat (LRR) domain of Nod2 have been implicated in the pathogenesis of Crohn's disease, yet the function of Nod2 and regulation of the Nod2 pathway remain unclear. In this study, we determined that mitogen-activated protein kinase kinase transforming growth factor (TGF)-beta-activated kinase 1 (TAK1) interacts with Nod2 and is required for Nod2-mediated NF-kappaB activation. The dominant negative form of TAK1 abolished muramyl dipeptide-induced NF-kappaB activation in Nod2-expressing cells. Nod2, acting in a reciprocal manner, inhibited TAK1-induced NF-kappaB activation in RICK-deficient embryonic fibroblasts. Nod2 appears to interact with TAK1 through its LRR region to exert its inhibitory effect on TAK1-induced NF-kappaB activation. Further, wild-type LRR more effectively suppressed NF-kappaB activation induced by TAK1 than LRR with a 3020insC mutation. Considered together, these findings demonstrate a critical role for TAK1 in Nod2-mediated innate immune responses and reveal a novel function for Nod2 in the regulation of the TAK1 signaling pathway.     

Leucine-rich repeat (LRR) proteins: Integrators of pattern recognition and signaling in immunity

The leucine-rich repeats (LRR)-containing domain is evolutionarily conserved in many proteins associated with innate immunity in plants, invertebrates and vertebrates. Serving as a first line of defense, the innate immune response is initiated through the sensing of pathogen-associated molecular patterns (PAMPs). In plants, NBS (nucleotide-binding site)-LRR proteins provide recognition of pathogen products of avirulence (AVR) genes. LRRs also promote interaction between LRR proteins as observed in receptor-coreceptor complexes. In mammals, toll-like receptors (TLRs) and NOD-like receptors (NLRs) through their LRR domain, sense molecular determinants from a structurally diverse set of bacterial, fungal, parasite and viral-derived components. In humans, at least 34 LRR proteins are implicated in diseases. Most LRR domains consist of 2–45 leucine-rich repeats, with each repeat about 20–30 residues long. Structurally, LRR domains adopt an arc or horseshoe shape, with the concave face consisting of parallel β-strands and the convex face representing a more variable region of secondary structures including helices. Apart from the TLRs and NLRs, most of the 375 human LRR proteins remain uncharacterized functionally. We incorporated computational and functional analyses to facilitate multifaceted insights into human LRR proteins and outline a few approaches here.     

The leucine-rich repeat domain in plant innate immunity: a wealth of possibilities

The innate immune system of both plants and animals uses immune receptors to detect pathogens and trigger defence responses. Despite having distinct evolutionary origin, most plant and animal immune receptors have a leucine-rich repeat (LRR) domain. The LRR domain adopts a slender conformation that maximizes surface area and has been shown to be ideal for mediating protein–protein interactions. Although the LRR domain was expected to be a platform for pathogen recognition, the NB-LRR class of plant innate immune receptors uses its LRR domain to carry out many other roles. This review discusses the domain architecture of plant LRRs and the various roles ascribed to this motif.     

Suprachiasmatic nucleus circadian oscillatory protein,a novel binding partner of K-Ras in the membrane rafts,negatively regulates MAPK pathway

Suprachiasmatic nucleus circadian oscillatory protein (SCOP) is a member of the leucine-rich repeat (LRR)-containing protein family. In addition to circadian expression in the rat hypothalamic suprachiasmatic nucleus, SCOP is constitutively expressed in neurons throughout the rat brain. Here we found that a substantial amount of SCOP was localized in the brain membrane rafts, in which only K-Ras was abundant among Ras isoforms. SCOP interacted directly through its LRR domain with a subset of K-Ras in the guanine nucleotide-free form that was present in the raft fraction. This interaction interfered with the binding of added guanine nucleotide to K-Ras in vitro. A negative regulatory role of SCOP for K-Ras function was examined in PC12 cell lines stably overexpressing SCOP or its deletion mutants. Overexpression of full-length SCOP markedly down-regulated ERK1/ERK2 activation induced by depolarization or phorbol ester stimulation, and this inhibitory effect of overexpressed SCOP was dependent on its LRR domain. These results strongly suggest that SCOP negatively regulates K-Ras signaling in the membrane rafts, identifying a novel mechanism for regulation of the Ras-MAPK pathway.     

Aromatic amino acids at the surface of InlB are essential for host cell invasion by Listeria monocytogenes

The surface protein InlB of the pathogen Listeria monocytogenes promotes invasion of this bacterium into host cells by binding to and activating the receptor tyrosine kinase Met. The curved leucine-rich repeat (LRR) domain of InlB, which is essential for this process, contains a string of five surface-exposed aromatic amino acid residues positioned along its concave face. Here, we show that the replacement of four of these residues (F104, W124, Y170 or Y214) by serine leads to a complete loss of uptake of latex beads coated with InlB', a truncated functional variant of InlB. The mutants correspondingly display severely reduced binding to Met. To abrogate fully invasion of bacteria expressing full-length InlB, exchange of at least four aromatic amino acids is required. We conclude that InlB binds to Met through its concave surface of the LRR domain, and that aromatic amino acids are critical for binding and signalling before invasion.     

The internal region leucine-rich repeat 6 of decorin interacts with low density lipoprotein receptor-related protein-1, modulates transforming growth factor (TGF)-β-dependent signaling, and inhibits TGF-β-dependent fibrotic response in skeletal muscles

Decorin is a small proteoglycan, composed of 12 leucine-rich repeats (LRRs) that modulates the activity of transforming growth factor type β (TGF-β) and other growth factors, and thereby influences proliferation and differentiation in a wide array of physiological and pathological processes, such as fibrosis, in several tissues and organs. Previously we described two novel modulators of the TGF-β-dependent signaling pathway: LDL receptor-related protein (LRP-1) and decorin. Here we have determined the regions in decorin that are responsible for interaction with LRP-1 and are involved in TGF-β-dependent binding and signaling. Specifically, we used decorin deletion mutants, as well as peptides derived from internal LRR regions, to determine the LRRs responsible for these decorin functions. Our results indicate that LRR6 and LRR5 participate in the interaction with LRP-1 and TGF-β as well as in its dependent signaling. Furthermore, the internal region (LRR6i), composed of 11 amino acids, is responsible for decorin binding to LRP-1 and subsequent TGF-β-dependent signaling. Furthermore, using an in vivo approach, we also demonstrate that the LRR6 region of decorin can inhibit TGF-β mediated action in response to skeletal muscle injury.     

Interaction between domains of a plant NBS-LRR protein in disease resistance-related cell death

Many plant disease resistance (R) genes encode proteins predicted to have an N-terminal coiled-coil (CC) domain, a central nucleotide-binding site (NBS) domain and a C-terminal leucine-rich repeat (LRR) domain. These CC-NBS-LRR proteins recognize specific pathogen-derived products and initiate a resistance response that often includes a type of cell death known as the hypersensitive response (HR). Co-expression of the potato CC-NBS-LRR protein Rx and its elicitor, the PVX coat protein (CP), results in a rapid HR. Surprisingly, co-expression of the LRR and CC-NBS as separate domains also resulted in a CP-dependent HR. Likewise, the CC domain complemented a version of Rx lacking this domain (NBS- LRR). Correspondingly, the LRR domain interacted physically in planta with the CC-NBS, as did CC with NBS-LRR. Both interactions were disrupted in the presence of CP. However, the interaction between CC and NBS-LRR was dependent on a wild-type P-loop motif, whereas the interaction between CC-NBS and LRR was not. We propose that activation of Rx entails sequential disruption of at least two intramolecular interactions.     

Expression, purification and preliminary biochemical and structural characterization of the leucine rich repeat namesake domain of leucine rich repeat kinase 2

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. Much research effort has been directed towards the catalytic core region of LRRK2 composed of GTPase (ROC, Ras of complex proteins) and kinase domains and a connecting COR (C-terminus of ROC) domain. In contrast, the precise functions of the protein-protein interaction domains, such as the leucine-rich repeat (LRR) domain, are not known. In the present study, we modeled the LRRK2 LRR domain (LRR(LRRK2)) using a template assembly approach, revealing the presence of 14 LRRs. Next, we focused on the expression and purification of LRR(LRRK2) in Escherichia coli. Buffer optimization revealed that the protein requires the presence of a zwitterionic detergent, namely Empigen BB, during solubilization and the subsequent purification and characterization steps. This indicates that the detergent captures the hydrophobic surface patches of LRR(LRRK2) thereby suppressing its aggregation. Circular dichroism (CD) spectroscopy measured 18% α-helices and 21% β-sheets, consistent with predictions from the homology model. Size exclusion chromatography (SEC) and dynamic light scattering measurements showed the presence of a single species, with a Stokes radius corresponding to the model dimensions of a protein monomer. Furthermore, no obvious LRR(LRRK2) multimerization was detected via cross-linking studies. Finally, the LRR(LRRK2) clinical mutations did not influence LRR(LRRK2) secondary, tertiary or quaternary structure as determined via SEC and CD spectroscopy. We therefore conclude that these mutations are likely to affect putative LRR(LRRK2) inter- and intramolecular interactions.     

Structural bases for recognition of Anp32/LANP proteins

The leucine-rich repeat acidic nuclear protein (Anp32a/LANP) belongs to a family of evolutionarily-conserved phosphoproteins involved in a complex network of protein-protein interactions. In an effort to understand the cellular role, we have investigated the mode of interaction of Anp32a with its partners. As a prerequisite, we solved the structure in solution of the evolutionarily conserved N-terminal leucine-rich repeat (LRR) domain and modeled its interactions with other proteins, taking PP2A as a paradigmatic example. The interaction between the Anp32a LRR domain and the AXH domain of ataxin-1 was probed experimentally. The two isolated and unmodified domains bind with very weak (millimolar) affinity, thus suggesting the necessity either for an additional partner (e.g. other regions of either or both proteins or a third molecule) or for a post-translational modification. Finally, we identified by two-hybrid screening a new partner of the LRR domain, i.e. the microtubule plus-end tracking protein Clip 170/Restin, known to regulate the dynamic properties of microtubules and to be associated with severe human pathologies.     

A mutation within the leucine-rich repeat domain of the Arabidopsis disease resistance gene RPS5 partially suppresses multiple bacterial and downy mildew resistance genes.

Recognition of pathogens by plants is mediated by several distinct families of functionally variable but structurally related disease resistance (R) genes. The largest family is defined by the presence of a putative nucleotide binding domain and 12 to 21 leucine-rich repeats (LRRs). The function of these LRRs has not been defined, but they are speculated to bind pathogen-derived ligands. We have isolated a mutation in the Arabidopsis RPS5 gene that indicates that the LRR region may interact with other plant proteins. The rps5-1 mutation causes a glutamate-to-lysine substitution in the third LRR and partially compromises the function of several R genes that confer bacterial and downy mildew resistance. The third LRR is relatively well conserved, and we speculate that it may interact with a signal transduction component shared by multiple R gene pathways.     

PLAP-1/asporin inhibits activation of BMP receptor via its leucine-rich repeat motif

We previously identified the novel gene, periodontal ligament-associated protein-1 (PLAP-1)/asporin and reported that PLAP-1/asporin inhibited bone morphogenetic protein-2 (BMP-2)-induced cytodifferentiation of periodontal ligament (PDL) cells probably by direct interaction with BMP-2. Here, we elucidated the detailed regulatory mechanism of this protein on BMP-2-induced cytodifferentiation of PDL cells. Recombinant PLAP-1/asporin inhibited BMP-2-induced cytodifferentiation of PDL cells and competitively prevented BMP-2 from binding to the BMP receptor-IB (BMPR-IB), resulting in inhibition of BMP-dependent activation of Smad proteins. The induction of mutation to the leucine-rich repeat (LRR) motif, especially LRR5, within PLAP-1/asporin rescued the inhibitory effect of PLAP-1/asporin on BMP-2. By contrast, a 26-amino acid peptide in the PLAP-1/asporin LRR5 sequence inhibited BMP-2 activity. Our findings indicate that PLAP-1/asporin inhibits BMP-2-induced differentiation of PDL cells resulting from inactivation of the BMP-2 signaling pathway and that LRR, especially LRR5 of PLAP-1/asporin, plays an important role in the PLAP-1/asporin-BMP-2 interaction.