The SAX-3 Receptor Stimulates Axon Outgrowth and the Signal Sequence and Transmembrane Domain Are Critical for SAX-3 Membrane Localization in the PDE Neuron of C. elegans

Despite the known importance of the transmembrane domain (TMD) of syndecan receptors in cell adhesion and signaling, the molecular basis for syndecan TMD function remains unknown. Using in vivo invertebrate models, we found that mammalian syndecan-2 rescued both the guidance defects in C. elegans hermaphrodite-specific neurons and the impaired development of the midline axons of Drosophila caused by the loss of endogenous syndecan. These compensatory effects, however, were reduced significantly when syndecan-2 dimerization-defective TMD mutants were introduced. To further investigate the role of the TMD, we generated a chimera, 2eTPC, comprising the TMD of syndecan-2 linked to the cytoplasmic domain of platelet-derived growth factor receptor (PDGFR). This chimera exhibited SDS-resistant dimer formation that was lost in the corresponding dimerization-defective syndecan-2 TMD mutant, 2eT(GL)PC. Moreover, 2eTPC specifically enhanced Tyr 579 and Tyr 857 phosphorylation in the PDGFR cytoplasmic domain, while the TMD mutant failed to support such phosphorylation. Finally, 2eTPC, but not 2eT(GL)PC, induced phosphorylation of Src and PI3 kinase (known downstream effectors of Tyr 579 phosphorylation) and promoted Src-mediated migration of NIH3T3 cells. Taken together, these data suggest that the TMD of a syndecan-2 specifically regulates receptor cytoplasmic domain function and subsequent downstream signaling events controlling cell behavior.

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Genetic Analysis of Caenorhabditis elegans glp-1 Mutants Suggests Receptor Interaction or Competition

Genetics ◽

10.1093/genetics/163.1.115 ◽

2003 ◽

Vol 163 (1) ◽

pp. 115-132 ◽

Cited By ~ 14

Author(s):

Anita S-R Pepper ◽

Darrell J Killian ◽

E Jane Albert Hubbard

Keyword(s):

Transmembrane Domain ◽

Limiting Factors ◽

Single Amino Acid ◽

Receptor Interaction ◽

Gain Of Function ◽

Ligand Interaction ◽

Receptor Proteins ◽

C Elegans ◽

Single Amino Acid Change ◽

Functional Defect

Abstract glp-1 encodes a member of the highly conserved LIN-12/Notch family of receptors that mediates the mitosis/meiosis decision in the C. elegans germline. We have characterized three mutations that represent a new genetic and phenotypic class of glp-1 mutants, glp-1(Pro). The glp-1(Pro) mutants display gain-of-function germline pattern defects, most notably a proximal proliferation (Pro) phenotype. Each of three glp-1(Pro) alleles encodes a single amino acid change in the extracellular part of the receptor: two in the LIN-12/Notch repeats (LNRs) and one between the LNRs and the transmembrane domain. Unlike other previously described gain-of-function mutations that affect this region of LIN-12/Notch family receptors, the genetic behavior of glp-1(Pro) alleles is not consistent with simple hypermorphic activity. Instead, the mutant phenotype is suppressed by wild-type doses of glp-1. Moreover, a trans-heterozygous combination of two highly penetrant glp-1(Pro) mutations is mutually suppressing. These results lend support to a model for a higher-order receptor complex and/or competition among receptor proteins for limiting factors that are required for proper regulation of receptor activity. Double-mutant analysis with suppressors and enhancers of lin-12 and glp-1 further suggests that the functional defect in glp-1(Pro) mutants occurs prior to or at the level of ligand interaction.

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The PBN1 Gene of Saccharomyces cerevisiae: An Essential Gene That Is Required for the Post-translational Processing of the Protease B Precursor

Genetics ◽

10.1093/genetics/149.3.1277 ◽

1998 ◽

Vol 149 (3) ◽

pp. 1277-1292 ◽

Cited By ~ 1

Author(s):

Rajesh R Naik ◽

Elizabeth W Jones

Keyword(s):

Saccharomyces Cerevisiae ◽

Secretory Pathway ◽

Transmembrane Domain ◽

Essential Gene ◽

Signal Sequence ◽

Signal Peptidase ◽

Amino Terminal ◽

Autocatalytic Cleavage ◽

Protease B ◽

Chromosome Iii

Abstract The vacuolar hydrolase protease B in Saccharomyces cerevisiae is synthesized as an inactive precursor (Prb1p). The precursor undergoes post-translational modifications while transiting the secretory pathway. In addition to N- and O -linked glycosylations, four proteolytic cleavages occur during the maturation of Prb1p. Removal of the signal peptide by signal peptidase and the autocatalytic cleavage of the large aminoterminal propeptide occur in the endoplasmic reticulum (ER). Two carboxy-terminal cleavages of the post regions occur in the vacuole: the first cleavage is catalyzed by protease A and the second results from autocatalysis. We have isolated a mutant, pbn1-1, that exhibits a defect in the ER processing of Prb1p. The autocatalytic cleavage of the propeptide from Prb1p does not occur and Prb1p is rapidly degraded in the cytosol. PBN1 was cloned and is identical to YCL052c on chromosome III. PBN1 is an essential gene that encodes a novel protein. Pbn1p is predicted to contain a sub-C-terminal transmembrane domain but no signal sequence. A functional HA epitope-tagged Pbn1p fusion localizes to the ER. Pbn1p is N-glycosylated in its amino-terminal domain, indicating a lumenal orientation despite the lack of a signal sequence. Based on these results, we propose that one of the functions of Pbn1p is to aid in the autocatalytic processing of Prb1p.

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Structure, function, and expression of SEL-1, a negative regulator of LIN-12 and GLP-1 in C. elegans

Development ◽

10.1242/dev.124.3.637 ◽

1997 ◽

Vol 124 (3) ◽

pp. 637-644 ◽

Cited By ~ 1

Author(s):

B. Grant ◽

I. Greenwald

Keyword(s):

Staining Pattern ◽

Signal Sequence ◽

Negative Regulator ◽

Yeast Gene ◽

Hydrophobic Region ◽

C Elegans ◽

Cell Ablation ◽

New Information ◽

Membrane Bound ◽

Punctate Staining

Previous work indicated that sel-1 functions as a negative regulator of lin-12 activity, and predicted that SEL-1 is a secreted or membrane associated protein. In this study, we describe cell ablation experiments that suggest sel-1 mutations elevate lin-12 activity cell autonomously. We also use transgenic approaches to demonstrate that the predicted signal sequence of SEL-1 can direct secretion and is important for function, while a C-terminal hydrophobic region is not required for SEL-1 function. In addition, by analyzing SEL-1 localization using specific antisera we find that SEL-1 is localized intracellularly, with a punctate staining pattern suggestive of membrane bound vesicles. We incorporate these observations, and new information about a related yeast gene, into a proposal for a possible mechanism for SEL-1 function in LIN-12 turnover.

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Identification of SERINC5-001 as the Predominant Spliced Isoform for HIV-1 Restriction

Journal of Virology ◽

10.1128/jvi.00137-17 ◽

2017 ◽

Vol 91 (10) ◽

Cited By ~ 23

Author(s):

Xianfeng Zhang ◽

Tao Zhou ◽

Jie Yang ◽

Yumei Lin ◽

Jing Shi ◽

...

Keyword(s):

Plasma Membrane ◽

Transmembrane Domain ◽

The Other ◽

Stable Expression ◽

Membrane Localization ◽

Plasma Membrane Localization ◽

Immunodeficiency Virus ◽

Alternatively Spliced ◽

Anti Hiv ◽

Hiv 1

ABSTRACT Among the five serine incorporator (SERINC) family members, SERINC5 (Ser5) was reported to strongly inhibit HIV-1 replication, which is counteracted by Nef. Ser5 produces 5 alternatively spliced isoforms: Ser5-001 has 10 putative transmembrane domains, whereas Ser5-004, -005, -008a, and -008b do not have the last one. Here, we confirmed the strong Ser5 anti-HIV-1 activity and investigated its isoforms' expression and antiviral activities. It was found that Ser5-001 transcripts were detected at least 10-fold more than the other isoforms by real-time quantitative PCR. When Ser5-001 and its two isoforms Ser5-005 and Ser5-008a were expressed from the same mammalian expression vector, only Ser5-001 was stably expressed, whereas the others were poorly expressed due to rapid degradation. In addition, unlike the other isoforms, which are located mainly in the cytoplasm, Ser5-001 is localized primarily to the plasma membrane. To map the critical determinant, Ser5 mutants bearing C-terminal deletions were created. It was found that the 10th transmembrane domain is required for Ser5 stable expression and plasma membrane localization. As expected, only Ser5-001 strongly inhibits HIV-1 infectivity, whereas the other Ser5 isoforms and mutants that do not have the 10th transmembrane domain show very poor activity. It was also observed that the Nef counteractive activity could be easily saturated by Ser5 overexpression. Thus, we conclude that Ser5-001 is the predominant antiviral isoform that restricts HIV-1, and the 10th transmembrane domain plays a critical role in this process by regulating its protein stability and plasma membrane targeting. IMPORTANCE Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express a small protein, Nef, to enhance viral pathogenesis in vivo. Nef has an important in vitro function, which is to make virus particles more infectious, but the mechanism has been unclear. Recently, Nef was reported to counteract a novel anti-HIV host protein, SERINC5 (Ser5). Ser5 has five alternatively spliced isoforms, Ser5-001, -004, -005, -008a, and -008b, and only Ser5-001 has an extra C-terminal transmembrane domain. We now show that the Ser5-001 transcripts are produced at least 10-fold more than the others, and only Ser5-001 produces stable proteins that are targeted to the plasma membrane. Importantly, only Ser5-001 shows strong anti-HIV-1 activity. We further demonstrate that the extra transmembrane domain is required for Ser5 stable expression and plasma membrane localization. These results suggest that plasma membrane localization is required for Ser5 antiviral activity, and Ser5-001 is the predominant isoform that contributes to the activity.

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Host-mediated RNAi of a Notch-like receptor gene inMeloidogyne incognitainduces nematode resistance

Parasitology ◽

10.1017/s0031182018000641 ◽

2018 ◽

Vol 145 (14) ◽

pp. 1896-1906

Author(s):

Deshika Kohli ◽

Parameswaran Chidambaranathan ◽

J. Prasanth Tej Kumar ◽

Ashish Kumar Singh ◽

Anil Kumar ◽

...

Keyword(s):

Transmembrane Domain ◽

Receptor Gene ◽

Nematode Resistance ◽

Receptor Protein ◽

Binding Motifs ◽

C Elegans ◽

Transgenic Lines ◽

Germline Proliferation ◽

Epidermal Growth ◽

Almost All

AbstractGLP-1 (abnormal germline proliferation) is a Notch-like receptor protein that plays an essential role in pharyngeal development. In this study, an orthologue ofCaenorhabditis elegans glp-1was identified inMeloidogyne incognita. A computational analysis revealed that the orthologue contained almost all the domains present in theC. elegansgene: specifically, the LIN-12/Notch repeat, the ankyrin repeat, a transmembrane domain and different ligand-binding motifs were present in orthologue, but the epidermal growth factor-like motif was not observed. An expression analysis showed differential expression ofglp-1throughout the life cycle ofM. incognita, with relatively higher expression in the egg stage. To evaluate the silencing efficacy ofMi-glp-1, transgenicArabidopsisplants carrying double-stranded RNA constructs ofglp-1were generated, and infection of these plants withM. incognitaresulted in a 47–50% reduction in the numbers of galls, females and egg masses. Females obtained from the transgenic RNAi lines exhibited 40–60% reductions in the transcript levels of the targetedglp-1gene compared with females isolated from the control plants. Second-generation juveniles (J2s), which were descendants of the infected females from the transgenic lines, showed aberrant phenotypes. These J2s exhibited a significant decrease in the overall distance from the stylet to the metacorpus region, and this effect was accompanied by disruption around the metacorporeal bulb of the pharynx. The present study suggests a role for this gene in organ (pharynx) development during embryogenesis inM. incognitaand its potential use as a target in the management of nematode infestations in plants.

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Monoclonal antibodies specific for low-affinity interleukin-3 (IL-3) binding protein AIC2A: evidence that AIC2A is a component of a high- affinity IL-3 receptor

Blood ◽

10.1182/blood.v79.4.895.895 ◽

1992 ◽

Vol 79 (4) ◽

pp. 895-903

Author(s):

T Ogorochi ◽

T Hara ◽

HM Wang ◽

K Maruyama ◽

A Miyajima

Keyword(s):

Monoclonal Antibodies ◽

Transmembrane Domain ◽

Signal Sequence ◽

Amino Acid Level ◽

Terminal Sequence ◽

Chimeric Receptors ◽

Interleukin 3 ◽

High Affinity ◽

Processing Site ◽

Binding Component

Abstract Mouse interleukin-3 (IL-3) binds to its receptor with high and low affinities. Using anti-Aic2 antibody, two distinct cDNAs (AIC2A and AIC2B) were isolated. The AIC2A gene encodes a protein of 120 Kd that binds IL-3 with low affinity, whereas the AIC2B gene encodes a protein that is 91% identical to AIC2A at the amino acid level, but which does not bind IL-3. To study the structure of the functional high-affinity IL-3 receptor (IL-3R), we generated specific monoclonal antibodies against the AIC2A protein. We produced a soluble AIC2A protein by inserting a termination codon at the beginning of the transmembrane domain of the AIC2A cDNA. Soluble AIC2A protein expressed in COS7 cells was purified to homogeneity and three anti-AIC2A monoclonal antibody- producing hybridomas (3D1, 3D4, and 9D3) were obtained from a rat immunized with the purified soluble AIC2A protein. The antibodies were specific for the AIC2A protein and did not bind to the AIC2B protein. Using chimeric receptors between AIC2A and AIC2B, recognition sites of the antibodies were mapped. The antibodies immunoprecipitated a 120-Kd protein from IL-3-dependent PT18 cells. The N-terminal sequence of the 120-Kd protein was consistent with the predicted processing site of the signal sequence of the AIC2A protein. Staining of IL-3-dependent and IL- 3-independent cell lines with the 9D3 antibody were consistent with the IL-3 binding. The 9D3 antibody inhibited both the high-affinity IL-3 binding and the low-affinity binding, as well as IL-3-dependent proliferation. These results indicate that the AIC2A protein is a binding component of a high-affinity IL-3R.

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