Lipid Raft Association of Carboxypeptidase E Is Necessary for Its Function as a Regulated Secretory Pathway Sorting Receptor

Carboxypeptidase E (CPE) functions as a regulated secretory pathway sorting receptor for several prohormones, including pro-opiomelanocortin (POMC), proenkephalin and proinsulin. The association of CPE with lipid rafts in the trans-Golgi network and secretory granule membranes is necessary for its sorting receptor function. We now provide evidence that a domain within the C-terminal 25 residues of CPE functions as a signal for both raft association and the sorting of CPE to the regulated secretory pathway. A fusion protein containing the extracellular domain of the human interleukin-2 receptor Tac (N-Tac) and the C-terminal 25 amino acids of CPE was transfected into Neuro2A cells. This fusion protein floated in sucrose density gradients, indicating raft association, and co-localized with chromogranin A (CGA), a secretory granule marker. To define further a minimum sequence required for raft association and sorting, deletion mutants of CPE that lacked the C-terminal four or 15 residues (CPE-Δ4 and CPE-Δ15 respectively) were transfected into a clone of CPE-deficient Neuro2A cells. In contrast with full-length CPE, neither CPE-Δ4 nor CPE-Δ15 floated in sucrose density gradients. The sorting of both CPE-Δ4 and CPE-Δ15 to the regulated secretory pathway was impaired, as indicated by significantly increased basal secretion and a lack of response to stimulation. Additionally, there was a significant decrease in the co-localization of mutant CPE immunofluorescence with CGA when compared with full-length CPE. Finally, the sorting of the prohormone POMC to the regulated pathway was impaired in cells transfected with either CPE-Δ4 or CPE-Δ15. We conclude that the sorting of CPE to the regulated secretory pathway in endocrine cells is mediated by lipid rafts, and that the C-terminal four residues of CPE, i.e. Thr431-Leu-Asn-Phe434, are required for raft association and sorting.

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Identification of a Novel Prohormone Sorting Signal-Binding Site on Carboxypeptidase E, a Regulated Secretory Pathway-Sorting Receptor

Molecular Endocrinology ◽

10.1210/mend.13.4.0267 ◽

1999 ◽

Vol 13 (4) ◽

pp. 527-536 ◽

Cited By ~ 19

Author(s):

Chun-Fa Zhang ◽

Christopher R. Snell ◽

Y. Peng Loh

Keyword(s):

Binding Site ◽

Secretory Pathway ◽

Sorting Signal ◽

Carboxypeptidase E ◽

Sorting Receptor ◽

Regulated Secretory Pathway

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The sorting of proglucagon to secretory granules is mediated by carboxypeptidase E and intrinsic sorting signals

Journal of Endocrinology ◽

10.1530/joe-12-0468 ◽

2013 ◽

Vol 217 (2) ◽

pp. 229-240 ◽

Cited By ~ 15

Author(s):

Rebecca McGirr ◽

Leonardo Guizzetti ◽

Savita Dhanvantari

Keyword(s):

Secretory Pathway ◽

Secretory Granules ◽

Alpha Helix ◽

Intestinal Cell ◽

Alpha Cells ◽

Pancreatic Alpha Cells ◽

Carboxypeptidase E ◽

L Cells ◽

Sorting Signals ◽

Regulated Secretory Pathway

Proglucagon is expressed in pancreatic alpha cells, intestinal L cells and brainstem neurons. Tissue-specific processing of proglucagon yields the peptide hormones glucagon in the alpha cell and glucagon-like peptide (GLP)-1 and GLP-2 in L cells. Both glucagon and GLP-1 are secreted in response to nutritional status and are critical for regulating glycaemia. The sorting of proglucagon to the dense-core secretory granules of the regulated secretory pathway is essential for the appropriate secretion of glucagon and GLP-1. We examined the roles of carboxypeptidase E (CPE), a prohormone sorting receptor, the processing enzymes PC1/3 and PC2 and putative intrinsic sorting signals in proglucagon sorting. In Neuro 2a cells that lacked CPE, PC1/3 and PC2, proglucagon co-localised with the Golgi marker p115 as determined by quantitative immunofluorescence microscopy. Expression of CPE, but not of PC1/3 or PC2, enhanced proglucagon sorting to granules. siRNA-mediated knockdown of CPE disrupted regulated secretion of glucagon from pancreatic-derived alphaTC1–6 cells, but not of GLP-1 from intestinal cell-derived GLUTag cells. Mutation of the PC cleavage site K70R71, the dibasic R17R18 site within glucagon or the alpha-helix of glucagon, all significantly affected the sub-cellular localisation of proglucagon. Protein modelling revealed that alpha helices corresponding to glucagon, GLP-1 and GLP-2, are arranged within a disordered structure, suggesting some flexibility in the sorting mechanism. We conclude that there are multiple mechanisms for sorting proglucagon to the regulated secretory pathway, including a role for CPE in pancreatic alpha cells, initial cleavage at K70R71 and multiple sorting signals.

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The pro region is not required for the expression or intracellular routeing of carboxypeptidase E

Biochemical Journal ◽

10.1042/bj3230265 ◽

1997 ◽

Vol 323 (1) ◽

pp. 265-271 ◽

Cited By ~ 11

Author(s):

Lixin SONG ◽

Lloyd D. FRICKER

Keyword(s):

Confocal Microscopy ◽

Secretory Pathway ◽

Secretory Vesicles ◽

Wild Type ◽

Carboxypeptidase E ◽

N Terminus ◽

Regulated Secretory Pathway ◽

Golgi Network ◽

Pro Region ◽

Stimulation Of

Carboxypeptidase E (CPE) is initially synthesized as a larger precursor containing an additional 14-residue propeptide that is highly conserved between human and rat. Previous studies have established that the proenzyme is enzymically active and that deletion of the pro region does not affect the expression of the active enzyme. In the present study the function of the pro region was examined both by deleting this region from CPE and by attaching this region to the N-terminus of albumin. CPE lacking the pro region is sorted into the regulated secretory pathway in AtT-20 cells, based on confocal microscopy and examination of the stimulated secretion of the protein. Stimulation of AtT-20 cells with either forskolin or phorbol 12-myristate 13-acetate induces the secretion of wild-type CPE and of CPE lacking the pro region to similar extents, indicating a similar efficiency of sorting of the mutant. When the pro region of proalbumin is replaced with the pro region of CPE followed by expression in AtT-20 cells, the protein is not sorted into the regulated pathway, based on the lack of stimulated secretion. Confocal microscopy suggests that the proCPE/albumin protein is retained in the endoplasmic reticulum to a greater extent than is proalbumin. Pulse-chase analysis indicates that the pro region of CPE is not efficiently removed from the N-terminus of albumin, and the small amount of propeptide cleavage that does occur takes place soon before secretion of the protein. In contrast, confocal microscopy indicates that the majority of the propeptide is removed from CPE, and that this cleavage occurs in the trans-Golgi network or soon after sorting into the secretory vesicles. Taken together, these results suggest that the pro region of CPE is not required for the expression or intracellular routeing of this protein.

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Recycling of Raft-associated Prohormone Sorting Receptor Carboxypeptidase E Requires Interaction with ARF6

Molecular Biology of the Cell ◽

10.1091/mbc.e02-11-0758 ◽

2003 ◽

Vol 14 (11) ◽

pp. 4448-4457 ◽

Cited By ~ 30

Author(s):

Irina Arnaoutova ◽

Catherine L. Jackson ◽

Omayma S. Al-Awar ◽

Julie G. Donaldson ◽

Y. Peng Loh

Keyword(s):

Plasma Membrane ◽

Lipid Raft ◽

Endocrine Cells ◽

Transmembrane Domain ◽

Cytoplasmic Tail ◽

Small Gtpase ◽

Α Subunit ◽

Carboxypeptidase E ◽

Early Endosomes ◽

Sorting Receptor

Little is known about the molecular mechanism of recycling of intracellular receptors and lipid raft-associated proteins. Here, we have investigated the recycling pathway and internalization mechanism of a transmembrane, lipid raft-associated intracellular prohormone sorting receptor, carboxypeptidase E (CPE). CPE is found in the trans-Golgi network (TGN) and secretory granules of (neuro)endocrine cells. An extracellular domain of the IL2 receptor α-subunit (Tac) fused to the transmembrane domain and cytoplasmic tail of CPE (Tac-CPE25) was used as a marker to track recycling of CPE. We show in (neuro)endocrine cells, that upon stimulated secretory granule exocytosis, raft-associated Tac-CPE25 was rapidly internalized from the plasma membrane in a clathrin-independent manner into early endosomes and then transported through the endocytic recycling compartment to the TGN. A yeast two-hybrid screen and in vitro binding assay identified the CPE cytoplasmic tail sequence S472ETLNF477 as an interactor with active small GTPase ADP-ribosylation factor (ARF) 6, but not ARF1. Expression of a dominant negative, inactive ARF6 mutant blocked this recycling. Mutation of residues S472 or E473 to A in the cytoplasmic tail of CPE obliterated its binding to ARF6, and internalization from the plasma membrane of Tac-CPE25 mutated at S472 or E473 was significantly reduced. Thus, CPE recycles back to the TGN by a novel mechanism requiring ARF6 interaction and activity.

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Carboxypeptidase E and Secretogranin III Coordinately Facilitate Efficient Sorting of Proopiomelanocortin to the Regulated Secretory Pathway in AtT20 Cells

Molecular Endocrinology ◽

10.1210/me.2015-1166 ◽

2016 ◽

Vol 30 (1) ◽

pp. 37-47 ◽

Cited By ~ 10

Author(s):

Niamh X. Cawley ◽

Trushar Rathod ◽

Sigrid Young ◽

Hong Lou ◽

Nigel Birch ◽

...

Keyword(s):

Secretory Pathway ◽

Carboxypeptidase E ◽

Regulated Secretory Pathway

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Dissecting carboxypeptidase E: properties, functions and pathophysiological roles in disease

Endocrine Connections ◽

10.1530/ec-17-0020 ◽

2017 ◽

Vol 6 (4) ◽

pp. R18-R38 ◽

Cited By ~ 15

Author(s):

Lin Ji ◽

Huan-Tong Wu ◽

Xiao-Yan Qin ◽

Rongfeng Lan

Keyword(s):

Bone Mineral Density ◽

Secretory Pathway ◽

Bioactive Peptides ◽

Vesicle Transport ◽

Mineral Density ◽

Truncated Form ◽

Carboxypeptidase E ◽

Wide Range ◽

Regulated Secretory Pathway ◽

Endocrine Tissues

Since discovery in 1982, carboxypeptidase E (CPE) has been shown to be involved in the biosynthesis of a wide range of neuropeptides and peptide hormones in endocrine tissues, and in the nervous system. This protein is produced from pro-CPE and exists in soluble and membrane forms. Membrane CPE mediates the targeting of prohormones to the regulated secretory pathway, while soluble CPE acts as an exopeptidase and cleaves C-terminal basic residues from peptide intermediates to generate bioactive peptides. CPE also participates in protein internalization, vesicle transport and regulation of signaling pathways. Therefore, in two types of CPE mutant mice, Cpefat/Cpefat and Cpe knockout, loss of normal CPE leads to a lot of disorders, including diabetes, hyperproinsulinemia, low bone mineral density and deficits in learning and memory. In addition, the potential roles of CPE and ΔN-CPE, an N-terminal truncated form, in tumorigenesis and diagnosis were also addressed. Herein, we focus on dissecting the pathophysiological roles of CPE in the endocrine and nervous systems, and related diseases.

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