The endocytic pathways of a secretory granule membrane protein in HEK293 cells: PAM and EGF traverse a dynamic multivesicular body network together

Back, Nathan; Kanerva, Kristiina; Kurutihalli, Vishwanatha; Yanik, Andrew; Ikonen, Elina; Mains, Richard E.; Eipper, Betty A.

doi:10.1016/j.ejcb.2017.03.007

Cited by 13 publications

(12 citation statements)

References 36 publications

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“…Internalized ligands either dissociate from receptors, such as LDL, or stay associated with receptors, such as EGF, inside LEs ( 43 ). Although co-localization between EGFR and PS-ASOs was observed, not all PS-ASOs were at EGFR-containing loci.…”

Section: Discussionmentioning

confidence: 99%

Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides

Wang

Allen

Vickers

et al. 2018

Nucleic Acids Research

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Chemically modified antisense oligonucleotides (ASOs) with phosphorothioate (PS) linkages have been extensively studied as research and therapeutic agents. PS-ASOs can enter the cell and trigger cleavage of complementary RNA by RNase H1 even in the absence of transfection reagent. A number of cell surface proteins have been identified that bind PS-ASOs and mediate their cellular uptake; however, the mechanisms that lead to productive internalization of PS-ASOs are not well understood. Here, we characterized the interaction between PS-ASOs and epidermal growth factor receptor (EGFR). We found that PS-ASOs trafficked together with EGF and EGFR into clathrin-coated pit structures. Their co-localization was also observed at early endosomes and inside enlarged late endosomes. Reduction of EGFR decreased PS-ASO activity without affecting EGF-mediated signaling pathways and overexpression of EGFR increased PS-ASO activity in cells. Furthermore, reduction of EGFR delays PS-ASO trafficking from early to late endosomes. Thus, EGFR binds to PS-ASOs at the cell surface and mediates essential steps for active (productive) cellular uptake of PS-ASOs through its cargo-dependent trafficking processes which migrate PS-ASOs from early to late endosomes. This EGFR-mediated process can also serve as an additional model to better understand the mechanism of intracellular uptake and endosomal release of PS-ASOs.

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Section: Discussionmentioning

confidence: 99%

Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides

Wang

Allen

Vickers

et al. 2018

Nucleic Acids Research

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“…Upon MVB fusion with the plasma membrane, exosomes are released. Vertebrate PAM and furin function in the endocytic pathway, with PAM identified in exosomes from several different sources [52,53]. Our data suggest that the controlled endoproteolytic activation of proneuropeptides and growth factors had their molecular and enzymatic origins in unicellular organisms.…”

Section: Discussionmentioning

confidence: 73%

Proteases Shape the Chlamydomonas Secretome: Comparison to Classical Neuropeptide Processing Machinery

et al. 2018

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The recent identification of catalytically active peptidylglycine α-amidating monooxygenase (PAM) in Chlamydomonas reinhardtii, a unicellular green alga, suggested the presence of a PAM-like gene and peptidergic signaling in the last eukaryotic common ancestor (LECA). We identified prototypical neuropeptide precursors and essential peptide processing enzymes (subtilisin-like prohormone convertases and carboxypeptidase B-like enzymes) in the C. reinhardtii genome. Reasoning that sexual reproduction by C. reinhardtii requires extensive communication between cells, we used mass spectrometry to identify proteins recovered from the soluble secretome of mating gametes, and searched for evidence that the putative peptidergic processing enzymes were functional. After fractionation by SDS-PAGE, signal peptide-containing proteins that remained intact, and those that had been subjected to cleavage, were identified. The C. reinhardtii mating secretome contained multiple matrix metalloproteinases, cysteine endopeptidases, and serine carboxypeptidases, along with one subtilisin-like proteinase. Published transcriptomic studies support a role for these proteases in sexual reproduction. Multiple extracellular matrix proteins (ECM) were identified in the secretome. Several pherophorins, ECM glycoproteins homologous to the Volvox sex-inducing pheromone, were present; most contained typical peptide processing sites, and many had been cleaved, generating stable N- or C-terminal fragments. Our data suggest that subtilisin endoproteases and matrix metalloproteinases similar to those important in vertebrate peptidergic and growth factor signaling play an important role in stage transitions during the life cycle of C. reinhardtii.

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“…Vegetative lytic enzyme 1 (VLE1), a subtilisin-like protease required for hatching, is released in daughter cell ectosomes [20], and the agglutinins essential for ciliary adhesion are present in ectosomes released by mating gametes [24,31]. Since mammalian PAM is found in the intraluminal vesicles of multivesicular bodies (MVBs) and in exosomes/extracellular vesicles purified from blood, saliva, and urine [32–35], we hypothesized that CrPAM would be released in ciliary ectosomes.…”

Section: Resultsmentioning

confidence: 99%

“…Mammalian PAM is present in the intraluminal vesicles of MVBs and in extracellular vesicles purified from blood, saliva, and urine [32–35]. With a multilayered cell wall covering all but its ciliary membrane, the origin of the bioactive ectosomes released by vegetative cells and adhering gametes is clear [20,22,24,31].…”

Section: Discussionmentioning

confidence: 99%

Cilia-based peptidergic signaling

et al. 2019

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Peptide-based intercellular communication is a ubiquitous and ancient process that predates evolution of the nervous system. Cilia are essential signaling centers that both receive information from the environment and secrete bioactive extracellular vesicles (ectosomes). However, the nature of these secreted signals and their biological functions remain poorly understood. Here, we report the developmentally regulated release of the peptide amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), and the presence of peptidergic signaling machinery (including propeptide precursors, subtilisin-like prohormone convertases, amidated products, and receptors) in ciliary ectosomes from the green alga Chlamydomonas. One identified amidated PAM product serves as a chemoattractant for mating-type minus gametes but repels plus gametes. Thus, cilia provide a previously unappreciated route for the secretion of amidated signaling peptides. Our study in Chlamydomonas and the presence of PAM in mammalian cilia suggest that ciliary ectosome-mediated peptidergic signaling dates to the early eukaryotes and plays key roles in metazoan physiology.

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The endocytic pathways of a secretory granule membrane protein in HEK293 cells: PAM and EGF traverse a dynamic multivesicular body network together

Cited by 13 publications

References 36 publications

Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides

Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides

Proteases Shape the Chlamydomonas Secretome: Comparison to Classical Neuropeptide Processing Machinery

Cilia-based peptidergic signaling

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