Distinct mechanisms orchestrate the contra-polarity of IRK and KOIN, two LRR-receptor-kinases controlling root cell division

In plants, cell polarity plays key roles in coordinating developmental processes. Despite the characterization of several polarly localized plasma membrane proteins, the mechanisms connecting protein dynamics with cellular functions often remain unclear. Here, we introduce a polarized receptor, KOIN, that restricts cell divisions in the Arabidopsis root meristem. In the endodermis, KOIN polarity is opposite to IRK, a receptor that represses endodermal cell divisions. Their contra-polar localization facilitates dissection of polarity mechanisms and the links between polarity and function. We find that IRK and KOIN are recognized, sorted, and secreted through distinct pathways. IRK extracellular domains determine its polarity and partially rescue the mutant phenotype, whereas KOIN’s extracellular domains are insufficient for polar sorting and function. Endodermal expression of an IRK/KOIN chimera generates non-cell-autonomous misregulation of root cell divisions that impacts patterning. Altogether, we reveal two contrasting mechanisms determining these receptors’ polarity and link their polarity to cell divisions in root tissue patterning.

The influence of the extracellular domains on the dynamic behavior of membrane proteins

The dynamic behavior of the plasma membrane proteins mediates various cellular processes, such as cell-cell interactions, transmembrane transport and signaling. It is widely accepted that the dynamics of the membrane proteins is determined either by the interactions of the transmembrane domain with the surrounding lipids or by the interaction of the intracellular domain with cytosolic components such as cortical actin. However, the impact of the extracellular domains (ECDs) on the dynamics of membrane proteins is rather unexplored. Here, we investigate how the ECD size influences protein dynamics in lipid bilayer. We reconstitute ECDs of different molecular weights and heights in model membrane systems and analyze ECD-driven protein sorting in lipid domains as well as protein mobility. We observe that increasing the ECD size leads to a decrease in ordered domain partitioning as well as diffusivity. Our data suggests a critical role of the ECDs on membrane protein behavior in the plasma membrane and paves the way to a more complete understanding of membrane protein dynamics that includes interaction with the extracellular matrix and glycocalyx in health and disease.

A designed fusion tag for soluble expression and selective separation of extracellular domains of fibroblast growth factor receptors

Fibroblast growth factor receptors (FGFRs) generate various transduction signals by interaction with fibroblast growth factors (FGFs) and are involved in various biological functions such as cell proliferation, migration, and differentiation. Malfunction of these proteins may lead to the development of various diseases, including cancer. Accordingly, FGFRs are considered an alternative therapeutic target for protein and/or gene therapy. However, the screening of antagonists or agonists of FGFRs is challenging due to their complex structural features associated with protein expression. Herein, we conducted the development of a protease-free cleavable tag (PFCT) for enhancing the solubility of difficult-to express protein by combining maltose-binding protein (MBP) and the C-terminal region of Npu intein. To validate the availability of the resulting tag for the functional production of extracellular domains of FGFRs (Ec_FGFRs), we performed fusion of PFCT with the N-terminus of Ec_FGFRs and analyzed the expression patterns. Almost all PFCT-Ec_FGFR fusion proteins were mainly detected in the soluble fraction except for Ec_FGFR4. Upon addition of the N-terminal region of Npu intein, approximately 85% of the PFCT-Ec_FGFRs was separated into PFCT and Ec_FGFR via intein-mediated cleavage. Additionally, the structural integrity of Ec_FGFR was confirmed by affinity purification using heparin column. Taken together, our study demonstrated that the PFCT could be used for soluble expression and selective separation of Ec_FGFRs.

Bifunctional Peptide that Anneals to Damaged Collagen and Clusters TGF-β Receptors Enhances Wound Healing

ABSTRACTTransforming growth factor-β (TGF-β) plays important roles in wound healing. The activity of TGF-β is initiated upon binding of the growth factor to extracellular domains of its receptors. We sought to facilitate activation by clustering these extracellular domains. To do so, we used a known peptide that binds to TGF-β receptors without diminishing their affinity for TGF-β. We conjugated this peptide to a collagen-mimetic peptide that can anneal to damaged collagen in a wound bed. We find that the conjugate enhances collagen deposition and wound closure in mice in a manner consistent with the clustering of TGF-β receptors. This strategy provides a means to upregulate the TGF-β signaling pathway without adding exogenous TGF-β and could inspire means to treat severe wounds.TOC Graphic

NLRR1 Is a Potential Therapeutic Target in Neuroblastoma and MYCN-Driven Malignant Cancers

Receptor tyrosine kinases (RTKs) receive different modulation before transmitting proliferative signals. We previously identified neuronal leucine-rich repeat 1 (NLRR1) as a positive regulator of EGF and IGF-1 signals in high-risk neuroblastoma cells. Here, we show that NLRR1 is up-regulated in various adult cancers and acts as a key regulator of tumor cell proliferation. In the extracellular domains of NLRR1, fibronectin type III (FNIII) domain is responsible for its function to promote cell proliferation. We generated monoclonal antibodies against the extracellular domains of NLRR1 (N1mAb) and screened the positive N1mAbs for growth inhibitory effect. The treatment of N1mAbs reduces tumor cell proliferation in vitro and in vivo, and sensitizes the cells to EGFR inhibitor, suggesting that NLRR1 is a novel regulatory molecule of RTK function. Importantly, epitope mapping analysis has revealed that N1mAbs with growth inhibitory effect recognize immunoglobulin-like and FNIII domains of NLRR1, which also indicates the importance of FNIII domain in the function of NLRR1. Thus, the present study provides a new insight into the development of a cancer therapy by targeting NLRR1 as a modulator of proliferative signals on cellular membrane of tumor cells.

The Extracellular Domains of GluN Subunits Play an Essential Role in Processing NMDA Receptors in the ER

N-methyl-D-aspartate receptors (NMDARs) belong to a family of ionotropic glutamate receptors that play essential roles in excitatory neurotransmission and synaptic plasticity in the mammalian central nervous system (CNS). Functional NMDARs consist of heterotetramers comprised of GluN1, GluN2A-D, and/or GluN3A-B subunits, each of which contains four membrane domains (M1 through M4), an intracellular C-terminal domain, a large extracellular N-terminal domain composed of the amino-terminal domain and the S1 segment of the ligand-binding domain (LBD), and an extracellular loop between M3 and M4, which contains the S2 segment of the LBD. Both the number and type of NMDARs expressed at the cell surface are regulated at several levels, including their translation and posttranslational maturation in the endoplasmic reticulum (ER), intracellular trafficking via the Golgi apparatus, lateral diffusion in the plasma membrane, and internalization and degradation. This review focuses on the roles played by the extracellular regions of GluN subunits in ER processing. Specifically, we discuss the presence of ER retention signals, the integrity of the LBD, and critical N-glycosylated sites and disulfide bridges within the NMDAR subunits, each of these steps must pass quality control in the ER in order to ensure that only correctly assembled NMDARs are released from the ER for subsequent processing and trafficking to the surface. Finally, we discuss the effect of pathogenic missense mutations within the extracellular domains of GluN subunits with respect to ER processing of NMDARs.

The Secreted Tyrosine Kinase Vlk Is Essential for Normal Platelet Activation and Thrombus Formation

Tyrosine phosphorylation of proteins secreted into the extracellular space has been observed in cell cultures and in vivo, yet little is known about the role that phosphorylation of extracellular proteins serves in modulating cell function. An important reason for the gap in our knowledge of the functional significance of extracellular protein phosphorylation has been the delay in identifying extracellular kinases. Within the last decade, however, bioinformatic strategies to identify kinases with signal peptides, coupled with biochemical approaches to characterize kinases in the secretory pathway, have described several kinases that phosphorylate secretory pathway and extracellular substrates. Of the known kinases containing signal sequences, Fam20B and VLK have been identified in platelets. VLK has been identified as a broadly expressed secretory pathway tyrosine kinase secreted from platelets in an activation dependent manner. Its role in platelet function, however, has not been previously studied. To understand the contribution of tyrosine phosphorylation of secreted factors and extracellular domains of transmembrane proteins in platelet function and thrombus formation, we generated mice whose platelets lacked VLK. Mice with megakaryocyte/platelet-specific VLK deficiency (Vlk-cKO) exhibited normal platelet abundance, volume and morphology, and tail clip bleeding times, but showed dramatic changes in platelet function in vitro and in vivo. In vivo, platelet accumulation was reduced by 90% in Vlk-cKO mice compared to control (Vlkf/f) littermates (P = 0.02) following laser-induced injury of cremaster arterioles (Figure). Likewise, fibrin generation was reduced in mice lacking platelet VLK by 62% (P = 0.009). In vitro, evaluation of resting and thrombin-stimulated VLK-deficient platelets demonstrated a significant decrease of several tyrosine phosphobands compared to control. Platelet function testing of VLK-deficient platelets (Figure) showed decreased platelet aggregation in response to stimulation with 100 µM AYPGKF, a PAR4 agonist, (Vlkf/f: 70+5.1%; Vlk-cKO: 23+8.0%) or 4 µg/mL collagen (Vlkf/f: 53+2.5%; Vlk-cKO: 27.5+2.9%). Dense and α-granule release in response to AYPGKF were also significantly decreased in platelets in which VLK had been silenced. In contrast, Vlk-cKO platelets aggregated normally in response to either 10 µM, 40 µM, or 100 µM ADP, and the aggregation defect in response to low doses of AYPGKF was reversed by subthreshold concentrations (2.5 µM) of ADP. Furthermore, stimulation with high-dose 150 µM AYPGFK or 5 U/ml thrombin resulted in comparable platelet function and ATP secretion in control and Vlk-cKO platelets respectively, ruling out a storage pool defect. Taken together, these results suggest that a dense granule secretion defect contributes to the decrease in platelet aggregation observed in platelets in which VLK is absent. In human platelets, tyrosines phosphorylated in secreted and extracellular domains of transmembrane proteins implicated in the regulation of platelet function were identified by mass spectroscopy analysis. Extracellular proteins or proteins with phosphosites that mapped to extracellular domains included ectonucleoside triphosphate diphosphodydrolase 6 [ENTPD6], platelet basic protein, integrin αIIß, and multimerin-1. These studies demonstrate that the secretory pathway tyrosine kinase VLK is critical for stimulus dependent platelet aggregation and thrombus formation, and provide the first evidence that secreted kinases contribute to platelet function. Disclosures De Ceunynck: Sanofi: Current Employment. Dilks:PlateletBiogenesis: Current Employment. Peters:PlateletBiogenesis: Current Employment. Noetzli:Anylam: Current Employment. Rosen:Keros Therapeutics: Membership on an entity's Board of Directors or advisory committees. Italiano:PlateletBioGenesis: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Flaumenhaft:STRM.Bio: Membership on an entity's Board of Directors or advisory committees; PlateletDiagnostics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; QuercisPharma: Membership on an entity's Board of Directors or advisory committees.