Y. Peng Loh

Carboxypeptidase E Is a Regulated Secretory Pathway Sorting Receptor: Genetic Obliteration Leads to Endocrine Disorders in Cpefat Mice

A proposed mechanism for sorting secretory proteins into granules for release via the regulated secretory pathway in endocrine-neuroendocrine cells involves binding the proteins to a sorting receptor at the trans-Golgi network, followed by budding and granule formation. We have identified such a sorting receptor as membrane-associated carboxypeptidase E (CPE) in pituitary Golgi-enriched and secretory granule membranes. CPE specifically bound regulated secretory pathway proteins, including prohormones, but not constitutively secreted proteins. We show that in the Cpe(fat) mutant mouse lacking CPE, the pituitary prohormone, pro-opiomelanocortin, was missorted to the constitutive pathway and secreted in an unregulated manner. Thus, obliteration of CPE, the sorting receptor, leads to multiple endocrine disorders in these genetically defective mice, including hyperproinsulinemia and infertility.

Sorting and Activity-Dependent Secretion of BDNF Require Interaction of a Specific Motif with the Sorting Receptor Carboxypeptidase E

Activity-dependent secretion of BDNF is important in mediating synaptic plasticity, but how it is achieved is unclear. Here we uncover a sorting motif receptor-mediated mechanism for regulated secretion of BDNF. X-ray crystal structure analysis revealed a putative sorting motif, I(16)E(18)I(105)D(106), in BDNF, which when mutated at the acidic residues resulted in missorting of proBDNF to the constitutive pathway in AtT-20 cells. A V20E mutation to complete a similar motif in NGF redirected a significant proportion of it from the constitutive to the regulated pathway. Modeling and binding studies showed interaction of the acidic residues in the BDNF motif with two basic residues in the sorting receptor, carboxypeptidase E (CPE). (35)S labeling experiments demonstrated that activity-dependent secretion of BDNF from cortical neurons was obliterated in CPE knockout mice. Thus, we have identified a mechanism whereby a specific motif I(16)E(18)I(105)D(106) interacts with CPE to sort proBDNF into regulated pathway vesicles for activity-dependent secretion.

The obesity susceptibility gene Cpe links FoxO1 signaling in hypothalamic pro-opiomelanocortin neurons with regulation of food intake

Reduced food intake brings about an adaptive decrease in energy expenditure that contributes to the recidivism of obesity after weight loss. Insulin and leptin inhibit food intake through actions in the central nervous system that are partly mediated by the transcription factor FoxO1. We show that FoxO1 ablation in pro-opiomelanocortin (Pomc)-expressing neurons in mice (here called Pomc-Foxo1−/− mice) increases Carboxypeptidase E (Cpe) expression, resulting in selective increases of α-melanocyte–stimulating hormone (α-Msh) and carboxy-cleaved β-endorphin, the products of Cpe-dependent processing of Pomc. This neuropeptide profile is associated with decreased food intake and normal energy expenditure in Pomc-Foxo1−/− mice. We show that Cpe expression is downregulated by diet-induced obesity and that FoxO1 deletion offsets the decrease, protecting against weight gain. Moreover, moderate Cpe overexpression in the arcuate nucleus phenocopies features of the FoxO1 mutation. The dissociation of food intake from energy expenditure in Pomc-Foxo1−/− mice represents a model for therapeutic intervention in obesity and raises the possibility of targeting Cpe to develop weight loss medications.

Lipid raft association of carboxypeptidase E is necessary for its function as a regulated secretory pathway sorting receptor

Membrane carboxypeptidase E (CPE) is a sorting receptor for targeting prohormones, such as pro-opiomelanocortin, to the regulated secretory pathway in endocrine cells. Its membrane association is necessary for it to bind a prohormone sorting signal at the trans-Golgi network (TGN) to facilitate targeting. In this study, we examined the lipid interaction of CPE in bovine pituitary secretory granule membranes, which are derived from the TGN. We show that CPE is associated with detergent-resistant lipid domains, or rafts, within secretory granule membranes. Lipid analysis revealed that these rafts are enriched in glycosphingolipids and cholesterol. Pulse-chase and subcellular fractionation experiments in AtT-20 cells show that the association of CPE with membrane rafts occurred only after it reached the Golgi. Cholesterol depletion resulted in dissociation of CPE from secretory granule membranes and decreased the binding of prohormones to membranes. In vivo cholesterol depletion using lovastatin resulted in the lack of sorting of CPE and its cargo to the regulated secretory pathway. We propose that the sorting receptor function of CPE necessitates its interaction with glycosphingolipid-cholesterol rafts at the TGN, thereby anchoring it in position to bind to its prohormone cargo.

CARBOXYPEPTIDASE E IS A SORTING RECEPTOR FOR PROHORMONES : BINDING AND KINETIC STUDIES

The binding of pro-opiomelanocortin,(POMC), pro-insulin, pro-enkephalin and chromogranin A (CGA) to the regulated secretory pathway sorting receptor, carboxypeptidase E (CPE), in bovine pituitary secretory granule (SG) membranes was investigated. N-POMC1-26, which contains the POMC sorting signal, bound to CPE in the SG membranes with low affinity and the binding was ion independent. Pro-insulin bound CPE with similar kinetics. Pro-enkephalin, but not CGA bound to CPE with similar IC50 as pro-insulin and N-POMC1-26. Crosslinking studies showed that pro-insulin and pro-enkephalin bound specifically to SG membrane CPE, similar to N-POMC1-26 reported previously. CPE was extracted from the SG membranes with NaHCO3 or KSCN, but not Triton X-100/1 M NaCl. The results show that CPE is tightly associated with SG membranes and binds several prohormones, but not CGA, with similar kinetics, providing further evidence that membrane CPE has the characteristics to function as a common sorting receptor for targeting prohormones to the regulated secretory pathway.

Chromogranin A Deficiency in Transgenic Mice Leads to Aberrant Chromaffin Granule Biogenesis

The biogenesis of dense-core secretory granules (DCGs), organelles responsible for the storage and secretion of neurotransmitters and neuropeptides in chromaffin cells, is poorly understood. Chromogranin A (CgA), which binds catecholamines for storage in the lumen of chromaffin granules, has been shown to be involved in DCG biogenesis in neuroendocrine PC12 cells. Here, we report that downregulation of CgA expression in vivo by expressing antisense RNA against CgA in transgenic mice led to a significant reduction in DCG formation in adrenal chromaffin cells. The number of DCGs formed in CgA antisense transgenic mice was directly correlated with the amount of CgA present in adrenal medulla. In addition, DCGs showed an increase in size, with enlargement in the volume around the dense core, a phenomenon that occurs to maintain constant “free” catecholamine concentration in the lumen of these granules. The extent of DCG swelling was inversely correlated with the number of DCGs formed, as well as the amount of CgA present in the adrenal glands of CgA antisense transgenic mice. These data indicate an essential role of CgA in regulating chromaffin DCG biogenesis and catecholamine storage in vivo.

An aminopeptidase activity in bovine pituitary secretory vesicles that cleaves the N-terminal arginine from β-lipotropin60–65

Secretory vesicles isolated from the neural and intermediate lobes of the bovine pituitary contained a membrane‐bound aminopeptidase activity which cleaved arginine from β‐LPH60–65 (Arg‐Tyr‐Gly‐Gly‐Phe‐Met) and Arg‐MCA. Neither methionine enkephalin (Tyr‐Gly‐Gly‐Phe‐Met) nor Substance P, which has an N‐terminal arginine followed by a proline, could serve as substrates for this aminopeptidase activity; nor could cathepsin B‐like or chymotrypsin‐like enzyme activities be detected in the vesicle preparations. Maximal enzyme activity was at pH 6.0, and the activity was inhibited by EDTA, stimulated by Co2+and Zn2+, but was unaffected by leupeptin, pepstatin A, phenylmethylsulfonyl fluoride and p‐chloromercuribenzenesulfonate, suggesting that the enzyme is a metalloaminopeptidase. The presence of this aminopeptidase activity in secretory vesicles suggests that it may be involved in peptide prohormone processing.

Pro‐Thyrotropin‐Releasing Hormone Processing by Recombinant PC1

Abstract: Pro‐thyrotropin‐releasing hormone (proTRH) is the precursor to thyrotropin‐releasing hormone (TRH; pGlu‐His‐Pro‐NH2), the hypothalamic releasing factor that stimulates synthesis and release of thyrotropin from the pituitary gland. Five copies of the TRH progenitor sequence (Gln‐His‐Pro‐Gly) and seven cryptic peptides are formed following posttranslational proteolytic cleavage of the 26‐kDa rat proTRH precursor. The endopeptidase(s) responsible for the physiological conversion of proTRH to the TRH progenitor form is currently unknown. We examined the in vitro processing of [3H]leucine‐labeled or unlabeled proTRH by partially purified recombinant PC1. Recombinant PC1 processed the 26‐kDa TRH precursor by initially cleaving the prohormone after the basic amino acid at either position 153 or 159. Based on the use of our well‐established antibodies, we propose that the initial cleavage gave rise to the formation of a 15‐kDa N‐terminal peptide (preproTRH25–152 or preproTRH25–158) and a 10‐kDa C‐terminal peptide (preproTRH154–255 or preproTRH160–255). Some initial cleavage occurred after amino acid 108 to generate a 16.5‐kDa C‐terminal peptide. The 15‐kDa N‐terminal intermediate was further processed to a 6‐kDa peptide (preproTRH25–76 or preproTRH25–82) and a 3.8‐kDa peptide (preproTRH83–108), whereas the 10‐kDa C‐terminal intermediate was processed to a 5.4‐kDa peptide (preproTRH206–255). The optimal pH for these cleavages was 5.5. ZnCl2, EDTA, EGTA, and the omission of Ca2+ inhibited the formation of pYE27 (preproTRH25–50), one of the proTRH N‐terminal products, by 48, 82, 72, and 45%, respectively. This study provides evidence, for the first time, that recombinant PC 1 enzyme can process proTRH to its predicted peptide intermediates.

Biosynthesis of Neuronal Peptides

The general acceptance of the idea that peptides represent a new class of intercellular messengers in the nervous system (i.e., neurotransmitters and neuromodulators) naturally raises the question whether the biosynthetic mechanisms for peptides in “peptidergic” neurons (i.e., neurons that synthesize peptides for release as intercellular messengers) confer distinct properties on these neurons. One of the central issues is whether neuronal peptides are synthesized by ribosomal mechanisms followed by posttranslational cleavage, or by enzymatic (“synthetase”) processes similar to those of the conventional neurotransmitters (e.g., acetylcholine, γ-aminobutyric acid, biogenic amines). The answer to this question has significance with regard to the cell biology and “functional—morphological” organization of these neurons. A ribosomal mechanism would imply that the biosynthetic process would be restricted to the neuronal perikaryon, whereas an enzymatic mechanism would allow biosynthesis and its regulation to occur at the neuron’s site of release in the axon terminal. In either case, axonal transport mechanisms would be intimately involved. In the former case, however, the transported material would be the presynthesized peptides, whereas in the latter, the biosynthetic enzyme would be transported.

Mechanism of sorting proopiomelanocortin and proenkephalin to the regulated secretory pathway of neuroendocrine cells.

Abstract: Proopiomelanocortin (POMC) and proenkephalin (PE) are synthesized at the endoplasmic reticulum and transported to the trans‐Golgi network (TGN) where they are sorted and packaged into dense‐core granules of the regulated secretory pathway (RSP). The mechanism of sorting POMC and PE to the RSP in neuroendocrine cells was investigated. Consensus sorting signals comprising two acidic residues and two hydrophobic residues exposed on the surface of N‐POMC1‐26 and N‐PE1‐32 were identified and shown to be sufficient and necessary for targeting POMC and PE to the RSP in PC12, Neuro2a, and AtT‐20 cells. The acidic residues of these sorting signals bind specifically to basic residues on the sorting receptor membrane, carboxypeptidase E (CPE), to effect sorting to the RSP. Analysis of POMC and PE sorting in Neuro2a cells depleted of CPE by CPE antisense RNA, and Cpefat/fat mouse pituitary cells lacking CPE showed missorting of both these molecules to the constitutive pathway in vivo. Thus, POMC and PE are sorted to the RSP at the TGN by a mechanism involving the interaction of a specific sorting signal on these molecules with the sorting receptor, CPE.