Variable Extracellular Domain Research Articles

Comparative analysis of the expression patterns of TM9SF family members in mice

Transmembrane 9 superfamily proteins (TM9SFs) define a highly conserved protein family, each member of which is characterized by a variable extracellular domain and presumably nine transmembrane domains. Although previous studies have delineated the potential cytological roles of TM9SFs like autophagy and secretory pathway, their functions during development are largely unknown. To establish the basis for dissecting the functions of TM9SFs in vivo, we employed the open-source database, structure prediction, immunofluorescence and Western blot to describe the gene and protein expression patterns of TM9SFs in human and mouse. While TM9SFs are ubiquitously and homogeneously expressed in all tissues in human with RNA sequencing and proteomics analysis, we found that all mice Tm9sf proteins are preferentially expressed in lung except Tm9sf1 which is enriched in brain although they all distributed in various tissues we examined. In addition, we further explored their expression patterns in the mice central nervous system (CNS) and its extension tissue retina. Interestingly, we could show that Tm9sf1is developmentally up-regulated in brain. In addition, we also detected all Tm9sf proteins are located in neurons and microglia instead of astrocytes. Importantly, Tm9sf3 is localized in the nuclei which is distinct from the other members that are dominantly targeted to the plasma membrane/cytoplasm as expected. Finally, we also found that Tm9sf family members are broadly expressed in the layers of INL, OPL, and GCL of retina and likely targeted to the plasma membrane of retinal cells. Thus, our data provided a comprehensive overview of TM9SFs expression patterns, illustrating their ubiquitous roles in different organs, implying the possible roles of Tm9sf2/3/4 in lung functions and Tm9sf1 in neurodevelopment, and highlighting a unique cell biological functions of TM9SF3 in neuronal and microglia.

An update on evolutionary, structural, and functional studies of receptor-like kinases in plants.

All living organisms must develop mechanisms to cope with and adapt to new environments. The transition of plants from aquatic to terrestrial environment provided new opportunities for them to exploit additional resources but made them vulnerable to harsh and ever-changing conditions. As such, the transmembrane receptor-like kinases (RLKs) have been extensively duplicated and expanded in land plants, increasing the number of RLKs in the advanced angiosperms, thus becoming one of the largest protein families in eukaryotes. The basic structure of the RLKs consists of a variable extracellular domain (ECD), a transmembrane domain (TM), and a conserved kinase domain (KD). Their variable ECDs can perceive various kinds of ligands that activate the conserved KD through a series of auto- and trans-phosphorylation events, allowing the KDs to keep the conserved kinase activities as a molecular switch that stabilizes their intracellular signaling cascades, possibly maintaining cellular homeostasis as their advantages in different environmental conditions. The RLK signaling mechanisms may require a coreceptor and other interactors, which ultimately leads to the control of various functions of growth and development, fertilization, and immunity. Therefore, the identification of new signaling mechanisms might offer a unique insight into the regulatory mechanism of RLKs in plant development and adaptations. Here, we give an overview update of recent advances in RLKs and their signaling mechanisms.

Kinase Function of Brassinosteroid Receptor Specified by Two Allosterically Regulated Subdomains.

Plants acquire the ability to adapt to the environment using transmembrane receptor-like kinases (RLKs) to sense the challenges from their surroundings and respond appropriately. RLKs perceive a variety of ligands through their variable extracellular domains (ECDs) that activate the highly conserved intracellular kinase domains (KDs) to control distinct biological functions through a well-developed downstream signaling cascade. A new study has emerged that brassinosteroid-insensitive 1 (BRI1) family and excess microsporocytes 1 (EMS1) but not GASSHO1 (GSO1) and other RLKs control distinct biological functions through the same signaling pathway, raising a question how the signaling pathway represented by BRI1 is specified. Here, we confirm that BRI1-KD is not functionally replaceable by GSO1-KD since the chimeric BRI1-GSO1 cannot rescue bri1 mutants. We then identify two subdomains S1 and S2. BRI1 with its S1 and S2 substituted by that of GSO1 cannot rescue bri1 mutants. Conversely, chimeric BRI1-GSO1 with its S1 and S2 substituted by that of BRI1 can rescue bri1 mutants, suggesting that S1 and S2 are the sufficient requirements to specify the signaling function of BRI1. Consequently, all the other subdomains in the KD of BRI1 are functionally replaceable by that of GSO1 although the in vitro kinase activities vary after replacements, suggesting their functional robustness and mutational plasticity with diverse kinase activity. Interestingly, S1 contains αC-β4 loop as an allosteric hotspot and S2 includes kinase activation loop, proposedly regulating kinase activities. Further analysis reveals that this specific function requires β4 and β5 in addition to αC-β4 loop in S1. We, therefore, suggest that BRI1 specifies its kinase function through an allosteric regulation of these two subdomains to control its distinct biological functions, providing a new insight into the kinase evolution.

The Crumbs3 Polarity Protein.

The Crumbs proteins are evolutionarily conserved apical transmembrane proteins. Drosophila Crumbs was discovered via its crucial role in epithelial polarity during fly embryogenesis. Crumbs proteins have variable extracellular domains but a highly conserved intracellular domain that can bind FERM and PDZ domain proteins. Mammals have three Crumbs genes and this review focuses on Crumbs3, the major Crumbs isoform expressed in mammalian epithelial cells. Although initial work has highlighted the role of Crumbs3 in polarity, more recent studies have found it has an important role in tissue morphogenesis functioning as a linker between the apical membrane and the actin cytoskeleton. In addition, recent publications have linked Crumbs3 to growth control via regulation of the Hippo/Yap pathway.

Structural Analysis of the Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) Intracellular Domain Reveals a Conserved Interaction Epitope

Plasmodium falciparum-infected red blood cells adhere to endothelial cells, thereby obstructing the microvasculature. Erythrocyte adherence is directly associated with severe malaria and increased disease lethality, and it is mediated by the PfEMP1 family. PfEMP1 clustering in knob-like protrusions on the erythrocyte membrane is critical for cytoadherence, however the molecular mechanisms behind this system remain elusive. Here, we show that the intracellular domains of the PfEMP1 family (ATS) share a unique molecular architecture, which comprises a minimal folded core and extensive flexible elements. A conserved flexible segment at the ATS center is minimally restrained by the folded core. Yeast-two-hybrid data and a novel sequence analysis method suggest that this central segment contains a conserved protein interaction epitope. Interestingly, ATS in solution fails to bind the parasite knob-associated histidine-rich protein (KAHRP), an essential cytoadherence component. Instead, we demonstrate that ATS associates with PFI1780w, a member of the Plasmodium helical interspersed sub-telomeric (PHIST) family. PHIST domains are widespread in exported parasite proteins, however this is the first specific molecular function assigned to any variant of this family. We propose that PHIST domains facilitate protein interactions, and that the conserved ATS epitope may be targeted to disrupt the parasite cytoadherence system.

Cyclic-nucleotide signalling in protozoa

Compared with the impressive progress in understanding signal transduction pathways and mechanisms in mammalian systems, advances in protozoan signalling processes, including cyclic nucleotide metabolism, have been very slow. This is in large part connected to the fact that the components of these pathways are very different in the protozoan parasites, as confirmed by the recently completed genome. For instance, kinetoplastids have no equivalents to the mammalian Class I adenylyl cyclases (ACs) in their genomes nor any of the subunits of the associated G-proteins. The cyclases in kinetoplastid parasites contain a single transmembrane domain, a conserved intracellular catalytic domain and a highly variable extracellular domain - consistent with the expression of multiple receptor-activated cyclases - but no receptor ligands, agonists or antagonists have been identified. Apicomplexan AC and guanylyl cyclase (GC) are even more unusual, potentially being bifunctional, harbouring either a putative ion channel (AC) or a P-type ATPase-like domain (GC) alongside the catalytic region. Phosphodiesterases (PDEs) and cyclic-nucleotide-activated protein kinases are essentially conserved in protozoa, although mostly insensitive to inhibitors of the mammalian proteins. Some of the PDEs have now been validated as promising drug targets. In the following manuscript, we will summarize the existing literature on cAMP and cGMP in protozoa: cyclases, PDEs and cyclic-nucleotide-dependent kinases.

Interactions between theS-Domain Receptor Kinases and AtPUB-ARM E3 Ubiquitin Ligases Suggest a Conserved Signaling Pathway in Arabidopsis

The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses.

Control of Cellular Physiology by TM9 Proteins in Yeast and Dictyostelium

TM9 proteins constitute a well defined family, characterized by the presence of a large variable extracellular domain and nine putative transmembrane domains. This family is highly conserved throughout evolution and comprises three members in Dictyostelium discoideum and Saccharomyces cerevisiae and four in humans and mice. In Dictyostelium, previous analysis demonstrated that TM9 proteins are implicated in cellular adhesion. In this study, we generated TM9 mutants in S. cerevisiae and analyzed their phenotype with particular attention to cellular adhesion. S. cerevisiae strains lacking any one of the three TM9 proteins were severely suppressed for adhesive growth and filamentous growth under conditions of nitrogen starvation. In these mutants, expression of the FLO11-lacZ reporter gene was strongly reduced, whereas expression of FRE(Ty1)-lacZ was not, suggesting that TM9 proteins are implicated at a late stage of nutrient-controlled signaling pathways. We also reexamined the phenotype of Dictyostelium TM9 mutant cells, focusing on nutrient-controlled cellular functions. Although the initiation of multicellular development and autophagy was unaltered in Dictyostelium TM9 mutants, nutrient-controlled secretion of lysosomal enzymes was dysregulated in these cells. These results suggest that in both yeast and amoebae, TM9 proteins participate in the control of specific cellular functions in response to changing nutrient conditions.

Changes in subcellular distribution of protocadherin gamma proteins accompany maturation of spinal neurons.

Protocadherins gamma (Pcdhgamma) are a family of transmembrane proteins in which variable extracellular domains are associated with an invariant cytoplasmic domain, potentially allowing these proteins to trigger common cellular responses through diverse extracellular signals. We studied the expression of the family by in situ hybridisation and immunohistochemistry for the conserved portion of the mRNA or protein. During mouse development, Pcdhgamma expression is highest in neural tissues, but is also present in some nonneural tissues. In the adult, Pcdhgamma expression is maintained at high levels in brain, in particular in hippocampus and in the Purkinje cells of the cerebellum, whereas it is downregulated in spinal cord. Using antibodies against the conserved cytoplasmic domain, we show that in cultured embryonic spinal cord neurons, Pcdhgamma protein is present initially in both axonal and dendritic growth cones. At later stages of differentiation in vitro, Pcdhgamma distribution becomes polarised to the somatodendritic compartment. We propose that members of the Pcdhgamma family may play roles in neuronal growth and maturation.

Ankyrin-binding proteins related to nervous system cell adhesion molecules: candidates to provide transmembrane and intercellular connections in adult brain.

A major class of ankyrin-binding glycoproteins have been identified in adult rat brain of 186, 155, and 140 kD that are alternatively spliced products of the same pre-mRNA. Characterization of cDNAs demonstrated that ankyrin-binding glycoproteins (ABGPs) share 72% amino acid sequence identity with chicken neurofascin, a membrane-spanning neural cell adhesion molecule in the Ig super-family expressed in embryonic brain. ABGP polypeptides have the following features consistent with a role as ankyrin-binding proteins in vitro and in vivo: (a) ABGPs and ankyrin associate as pure proteins in a 1:1 molar stoichiometry; (b) the ankyrin-binding site is located in the COOH-terminal 21 kD of ABGP186 which contains the predicted cytoplasmic domain; (c) ABGP186 is expressed at approximately the same levels as ankyrin (15 pmoles/milligram of membrane protein); and (d) ABGP polypeptides are co-expressed with the adult form of ankyrinB late in postnatal development and are colocalized with ankyrinB by immunofluorescence. Similarity in amino acid sequence and conservation of sites of alternative splicing indicate that genes encoding ABGPs and neurofascin share a common ancestor. However, the major differences in developmental expression reported for neurofascin in embryos versus the late postnatal expression of ABGPs suggest that ABGPs and neurofascin represent products of gene duplication events that have subsequently evolved in parallel with distinct roles. The predicted cytoplasmic domains of rat ABGPs and chicken neurofascin are nearly identical to each other and closely related to a group of nervous system cell adhesion molecules with variable extracellular domains, which includes L1, Nr-CAM, and Ng-CAM of vertebrates, and neuroglian of Drosophila. The ankyrin-binding site of rat ABGPs is localized to the C-terminal 200 residues which encompass the cytoplasmic domain, suggesting the hypothesis that ability to associate with ankyrin may be a shared feature of neurofascin and related nervous system cell adhesion molecules.