Proteolytic and cellular mechanisms in prohormone and proprotein processing 1570595534

M OLECULAR BIOLOGY I N T E L L I G E N C E U N I T

2

Vivian Y.H. Hook

Proteolytic and Cellular
Mechanisms in Prohormone
and Proprotein Processing

R.G. LANDES
C O M P A N Y


MOLECULAR BIOLOGY
INTELLIGENCE
UNIT

Proteolytic and Cellular
Mechanisms in Prohormone
and Proprotein Processing
Vivian Y.H. Hook
Department of Medicine
University of California, San Diego
La Jolla, California, U.S.A.

R.G. LANDES
COMPANY
AUSTIN, TEXAS
U.S.A.

MOLECULAR BIOLOGY INTELLIGENCE UNIT
Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing
R.G. LANDES COMPANY
Austin, Texas, U.S.A.
Copyright © 1998 R.G. Landes Company
All rights reserved.
No part of this book may be reproduced or transmitted in any form or by any means,
electronic or mechanical, including photocopy, recording, or any information storage and
retrieval system, without permission in writing from the publisher.
Printed in the U.S.A.
Please address all inquiries to the Publishers:
R.G. Landes Company, 810 South Church Street, Georgetown, Texas, U.S.A. 78626
Phone: 512/ 863 7762; FAX: 512/ 863 0081

ISBN: 1-57059-553-4

While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current
recommendations and practice at the time of publication, they make no warranty, expressed or
implied, with respect to material described in this book. In view of the ongoing research, equipment
development, changes in governmental regulations and the rapid accumulation of information
relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data

Proteolytic and cellular mechanisms in prohormone processing / [edited by] Vivian
Y.H. Hook.
p. cm. -- (Molecular biology intelligence unit)
ISBN 1-57059-553-4 (alk. paper)
1. Peptide hormones--Metabolism. 2. Proteolytic enzymes. 3. Peptide hormones-Physiological transport. 4. Protein precursors. 5. Post-translational modifications.

I. Hook, Vivian Yuan-Hen Ho, 1953- II. Series.
QP572.P4P767 1998
572'.76--dc21
98-28730
CIP


PUBLISHER’S NOTE
Landes Bioscience produces books in six Intelligence Unit series:
Medical, Molecular Biology, Neuroscience, Tissue Engineering,
Biotechnology and Environmental. The authors of our books are
acknowledged leaders in their fields. Topics are unique; almost
without exception, no similar books exist on these topics.
Our goal is to publish books in important and rapidly changing
areas of bioscience for sophisticated researchers and clinicians. To
achieve this goal, we have accelerated our publishing program to
conform to the fast pace at which information grows in bioscience.
Most of our books are published within 90 to 120 days of receipt of
the manuscript. We would like to thank our readers for their
continuing interest and welcome any comments or suggestions they
may have for future books.
Judith Kemper
Production Manager
R.G. Landes Company



CONTENTS
1. Targeting and Activation of Peptide Hormones
in the Secretory Pathway ........................................................................... 1

Ken Teter and Hsiao-Ping H. Moore
Introduction ............................................................................................. 1
Trafficking and Modification of Peptide Hormone Precursors ........... 3
Prohormone Sorting Mechanisms ......................................................... 8
Site of Prohormone Sorting .................................................................. 10
Prohormone Activation ........................................................................ 12
Summary and Future Perspectives ....................................................... 15
2. The Mechanism of Sorting Proopiomelanocortin to Secretory
Granules and Its Processing by Aspartic and PC Enzymes................... 29
Niamh X. Cawley, David R. Cool, Emmanuel Normant,
Fu-Sheng Shen, Vicki Olsen and Y. Peng Loh
General Introduction ............................................................................ 29
Mechanism of Sorting POMC
to the Regulated Secretory Pathway ................................................. 31
Endoproteolytic Processing of Proopiomelanocortin ......................... 34
Future Directions ................................................................................... 42
3. The Mammalian Precursor Convertases: Paralogs of the Subtilisin/
Kexin Family of Calcium-Dependent Serine Proteinases ..................... 49
Nabil G. Seidah, Majambu Mbikay, Mieczyslaw Marcinkiewicz,
Michel Chrétien
Introduction ........................................................................................... 49
Subtilisin/Kexin-like Precursor Convertases (PCs):
Structural and Cellular Considerations ............................................ 51
Ontogeny, Tissue Expression and Subcellular Localization ................ 59
Structure, Loci, and Evolution of PC Genes ........................................ 62
Antisense Transgene Inhibition ............................................................ 63
Heritable Deficiency of PC in Human and Mouse .............................. 65
Inhibitors of PCs .................................................................................... 66
Enzymatic Cascades: ADAM Family and PCs ..................................... 67
Conclusions ........................................................................................... 68

4. The Neuroendocrine Prohormone Convertases PC1, PC2 and PC5 ... 77
Margery C. Beinfeld
Introduction ........................................................................................... 77
The Discovery of the Subtilisin Family
of Prohormone Convertases ............................................................. 77
Distribution of PC1, PC2 and PC5 ....................................................... 79
Biosynthesis and Activation of PC1, PC2 and PC5 ............................. 79
Regulation of PC Expression ................................................................ 80
Experimental Systems Used to Study Processing ................................ 80
Enzymatic Activity of PC1, PC2, and PC5 ........................................... 81


Antisense PC1 and PC2 Strategies
to Study Proneuropeptide Processing ..............................................
Endoproteases in CCK Processing, a Case in Point .............................
Processing Enzyme Knockouts and Mutations ...................................
Future Challenges ..................................................................................

81
82
82
83

5. ‘Prohormone Thiol Protease’ (PTP), a Novel Cysteine Protease
for Proenkephalin and Prohormone Processing ................................... 89
Vivian Y.H. Hook, Yuan-Hsu Kang, Martin Schiller,
Nikolaos Tezapsidis, Jane M. Johnston and Ada Azaryan
Introduction ........................................................................................... 89
The Novel ‘Prohormone Thiol Protease’ (PTP): A Major
Proenkephalin Processing Enzyme in Chromaffin Granules ......... 92

Participation of PC1/3 and PC2 Subtilisin-Like Proteases,
and 70 kDa Aspartyl Protease (PCE) in Proenkephalin
Processing in Chromaffin Granules ............................................... 100
Conclusions ......................................................................................... 100
6. Regulation of Prohormone Conversion by Coordinated Control
of Processing Endopeptidase Biosynthesis with That
of the Prohormone Substrate ............................................................... 105
Terence P. Herbert, Cristina Alarcon, Robert H. Skelly,
L. Cornelius Bollheimer, George T. Schuppin and Christopher J. Rhodes
Introduction ......................................................................................... 105
Coordinated Regulation of Prohormone
and Processing Enzyme mRNA Levels ........................................... 106
Coordinated Translational Regulation of Specific Prohormone
and Processing Enzyme Biosynthesis ............................................. 110
7. Carboxypeptidase and Aminopeptidase Proteases
in Proneuropeptide Processing ............................................................ 121
Vivian Y.H. Hook and Sukkid Yasothornsrikul
Introduction ......................................................................................... 121
Neuroendocrine-specific Carboxypeptidase E/H .............................. 122
Molecular Genetic Analysis of Mutant Carboxypeptidase
E/H in fat/fat Obese Mice: Effects of Inactive CPE/H
on Prohormone Processing ............................................................ 129
Mutant CPE/H in fat/fat Mice Leads to Discovery
of Novel Carboxypeptidase D and Carboxypeptidase Z ............... 130
Evidence for CPE/H as a Sorting Receptor for the Intracellular
Routing of POMC and Possibly Other Prohormones
to the Secretory Vesicle ................................................................... 132
Aminopeptidase(s) for Prohormone Processing ............................... 133
Conclusions and Future Perspectives ................................................. 134

8. The Neuroendocrine Polypeptide 7B2 as a Molecular Chaperone
and Naturally Occurring Inhibitor
of Prohormone Convertase PC2 .......................................................... 141
A. Martin Van Horssen and Gerard J.M. Martens
Introduction ......................................................................................... 141
History of 7B2 ...................................................................................... 141
The 7B2 Gene and Its Regulation ....................................................... 142
Evolutionary Aspects ........................................................................... 144
7B2 is a Neuroendocrine-Specific Polypeptide .................................. 144
Biochemical Characteristics of 7B2 .................................................... 145
Posttranslational Modifications of 7B2 .............................................. 145
Regulated Secretion of 7B2 ................................................................. 146
The Quest for the Role of 7B2 ............................................................. 146
Model of the Interaction Between 7B2 and PC2 ............................... 149
Implications and Future Prospects ..................................................... 151
9. Neuroendocrine α1-Antichymotrypsin as a Possible Regulator
of Prohormone and Neuropeptide Precursor Processing .................. 159
Shin-Rong Hwang and Vivian Y.H. Hook
Introduction ......................................................................................... 159
Biochemical Evidence for α1-Antichymotrypsin (ACT) as an
Endogenous Regulator of the ‘Prohormone Thiol Protease’
(PTP) and Other Prohormone Processing Proteases .................... 160
Molecular Cloning Reveals Multiple Isoforms of Bovine ACT
Expressed in Neuroendocrine Tissues ........................................... 164
10. Proteolytic Inactivation of Secreted Neuropeptides ........................... 173
Eva Csuhai, Afshin Safavi, Michael W. Thompson and Louis B. Hersh
Introduction ......................................................................................... 173
Neprilysin ............................................................................................. 174
Aminopeptidases ................................................................................. 176

Angiotensin Converting Enzyme ........................................................ 178
Pyroglutamyl Peptidase II ................................................................... 178
Proline Specific Peptidases .................................................................. 179
Soluble Neuropeptidases ..................................................................... 180
Endopeptidase 24.15 and Endopeptidase 24.16. ................................ 181
Summary .............................................................................................. 182
11. Stimulation of Peptidergic Receptors by Peptide Hormones
and Neurotransmitters: Studies of Opioid Receptors ......................... 191
George Bot, Allan D. Blake and Terry Reisine
Introduction ......................................................................................... 191
Opioid Receptor Types ........................................................................ 191
Endogenous Opioids ........................................................................... 192
Endogenous Peptide Receptor Selectivity .......................................... 192
Opioid Ligands .................................................................................... 194


Opioid Cellular Activity ...................................................................... 195
Opioid Receptor Cloning .................................................................... 196
ORL1 and Nociceptin/Orphanin FQ .................................................. 196
Structure-Function Analysis of Cloned Opioid Receptors ............... 198
µ Receptor Knockout Mice Model ..................................................... 201
Agonist Regulation of Cloned Opioid Receptors .............................. 201
G Protein Role in Differential Agonist Activity ................................. 204
Conclusion ........................................................................................... 204
Index ................................................................................................................ 213


EDITORS
Vivian Y.H. Hook
Department of Medicine

University of California, San Diego
La Jolla, California, U.S.A.
Chapters 5, 7, 9

CONTRIBUTORS
Cristina Alarcon
Gifford Laboratories
for Diabetes Research
Department of of Internal Medicine
and Pharmacology
University of Texas
Southwestern Medical Center
Dallas, Texas, U.S.A.
Chapter 6

Cornelius Bollheimer
Gifford Laboratories
for Diabetes Research
Department of of Internal Medicine
and Pharmacology
University of Texas
Southwestern Medical Center
Dallas, Texas, U.S.A.
Chapter 6

Ada Azaryan
Department of Pharmacology
Uniformed Services
University of the Health Sciences
Bethesda, Maryland, U.S.A.

Chapter 5

George Bot
Department of Pharmacology
University of Pennsylvania
School of Medicine
Philadelphia, Pennsylvania, U.S.A.
Chapter 11

Margery C. Beinfeld
Department of Pharmacology
and Experimental Therapeutics
Tufts University School of Medicine
Boston, Massachusetts, U.S.A.
Chapter 4

Niamh X. Cawley
Section on Cellular Neurobiology
Laboratory of Developmental
Neurobiology
National Institutes of Child Health
and Human Development
National Institutes of Health
Bethesda, Maryland, U.S.A.
Chapter 2

Allan D. Blake
Department of Pharmacology
University of Pennsylvania
School of Medicine

Philadelphia, Pennsylvania, U.S.A.
Chapter 11

Michel Chrétien
J.A. De Sève Laboratories
of Molecular Neuroendocrinology
Clinical Research Institute of Montreal
Montreal, Quebec, Canada
Chapter 3


David R. Cool
Section on Cellular Neurobiology
Laboratory of Developmental
Neurobiology
National Institutes of Child Health
and Human Development
National Institutes of Health
Bethesda, Maryland, U.S.A.
Chapter 2
Eva Csuhai
Department of Biochemistry
College of Medicine
University of Kentucky
Lexington, Kentucky, U.S.A.
Chapter 10
Terence P. Herbert
Gifford Laboratories
for Diabetes Research
Department of of Internal Medicine

and Pharmacology
University of Texas
Southwestern Medical Center
Dallas, Texas, U.S.A.
Chapter 6
Louis B. Hersh
Department of Biochemistry
College of Medicine
University of Kentucky
Lexington, Kentucky, U.S.A.
Chapter 10
Shin-Rong Hwang
Department of Medicine
University of California, San Diego
La Jolla, California, U.S.A.
Chapter 9
Jane M. Johnston
Department of Neurological Surgery
Albert Einstein College of Medicine
Bronx, New York, U.S.A.
Chapter 5

Yuan-Hsu Kang
Naval Medical Research Institute
Bethesda, Maryland, U.S.A.
Chapter 5
Y. Peng Loh
Section on Cellular Neurobiology
Laboratory of Developmental
Neurobiology

National Institutes of Child Health
and Human Development
National Institutes of Health
Bethesda, Maryland, U.S.A.
Chapter 2
Mieczyslaw Marcinkiewicz
J.A. De Sève Laboratories
of Molecular Neuroendocrinology
Clinical Research Institute of Montreal
Montreal, Quebec, Canada
Chapter 3
Gerard J.M. Martens
Department of Animal Physiology
University of Nijmegen, Nijmegen
Toernooiveld, The Netherlands
Chapter 8
Majambu Mbikay
J.A. De Sève Laboratories
of Molecular Neuroendocrinology
Clinical Research Institute of Montreal
Montreal, Quebec, Canada
Chapter 3
Hsiao-Ping H. Moore
University of California at Berkeley
Department of Molecular
and Cell Biology
Berkeley, California, U.S.A.
Chapter 1

Emmanuel Normant
Section on Cellular Neurobiology
Laboratory of Developmental
Neurobiology
National Institutes of Child Health
and Human Development
National Institutes of Health
Bethesda, Maryland, U.S.A.
Chapter 2
Vicki Olsen
Section on Cellular Neurobiology
Laboratory of Developmental
Neurobiology
National Institutes of Child Health
and Human Development
National Institutes of Health
Bethesda, Maryland, U.S.A.
Chapter 2
Terry Reisine
Department of Pharmacology
University of Pennsylvania
School of Medicine
Philadelphia, Pennsylvania, U.S.A.
Chapter 11
Christopher J. Rhodes
Gifford Laboratories
for Diabetes Research
Department of of Internal Medicine
and Pharmacology
University of Texas

Southwestern Medical Center
Dallas, Texas, U.S.A.
Chapter 6
Afshin Safavi
Department of Biochemistry
College of Medicine
University of Kentucky
Lexington, Kentucky, U.S.A.
Chapter 10

Martin Schiller
Department of Neuroscience
Johns Hopkins University
Baltimore, Maryland, U.S.A.
Chapter 5
George T. Schuppin
Gifford Laboratories
for Diabetes Research
Department of of Internal Medicine
and Pharmacology
University of Texas
Southwestern Medical Center
Dallas, Texas, U.S.A.
Chapter 6
Nabil G. Seidah
J.A. De Sève Laboratories
of Biochemical Neuroendocrinology
Clinical Research Institute of Montreal
Montreal, Quebec, Canada
Chapter 3

Fu-Sheng Shen
Section on Cellular Neurobiology
Laboratory of Developmental
Neurobiology
National Institutes of Child Health
and Human Development
National Institutes of Health
Bethesda, Maryland, U.S.A.
Chapter 2
Robert H. Skelly
Gifford Laboratories
for Diabetes Research
Department of of Internal Medicine
and Pharmacology
University of Texas
Southwestern Medical Center
Dallas, Texas, U.S.A.
Chapter 6


Ken Teter
University of California at Berkeley
Department of Molecular
and Cell Biology
Berkeley, California, U.S.A.
Chapter 1
Nikolaos Tezapsidis
Department of Psychiatry
Mt. Sinai Medical Center
New York, New York, U.S.A.

Chapter 5
Michael W. Thompson
Department of Biochemistry
College of Medicine
University of Kentucky
Lexington, Kentucky, U.S.A.
Chapter 10

A. Martin Van Horssen
Department of Animal Physiology
University of Nijmegen, Nijmegen
Toernooiveld, The Netherlands
Chapter 8
Sukkid Yasothornsrikul
Department of Medicine
University of California, San Diego
La Jolla, California, U.S.A.
Chapter 7


PREFACE

P

roteolysis of proneuropeptides is key for the production of bioactive neuropeptides that mediate cell-cell communication in the endocrine and nervous systems. The conversion of inactive precursor to active peptide hormone
or neurotransmitter is required for neuroendocrine functions. This text is designed to provide the reader with an understanding of current knowledge concerning proteases involved in prohormone processing, and cellular aspects that
must be considered for proper processing, storage, and secretion of bioactive
peptides.
It is well known that prohormone processing occurs in well-defined subcellular compartments of the regulated secretory pathway. Knowledge of the
cell biology of prohormone processing is required in the search for processing

enzymes that are colocalized with prohormone substrates and neuropeptide
products. Therefore, important cellular aspects of the targeting and activation
of peptide hormones in the secretory pathway are discussed in the first two
chapters.
The next several chapters (chapters 2-6) present evidence for endoproteases
that have been demonstrated to be involved in prohormone processing. These
endoproteases include the large family of subtilisin/kexin prohormone
convertases, a novel cysteine protease known as ‘prohormone thiol protease’
(PTP), and an aspartyl protease that has been termed “POMC converting enzyme” (PCE). These studies provide evidence for three different mechanistic
classes of endoproteases that participate in prohormone processing. Subsequent
to the actions of endoproteases, carboxypeptidase and aminopeptidase enzymes
(discussed in chapter 7) that remove basic amino acids from the COOH- and
NH2-termini of peptide intermediates are needed to complete the proteolytic
processing of precursors into peptide forms. Moreover, recent molecular genetic studies illustrate the role of prohormone convertases 1 and 2, as well as
the carboxypeptidase E/H, in obesity and conditions related to diabetes.
The processing pathway is critical for generating active peptides. Therefore, it is likely that endogenous regulators exist that control the prohormone
processing pathway. Evidence for the 7B2 polypeptide as a molecular chaperone and inhibitor of a prohormone convertase is discussed in chapter 8. Also,
the role of endogenous isoforms of the protease inhibitor α1-antichymotrypsin
in regulating prohormone processing enzymes is presented in chapter 9.
Upon secretion of neuropeptides into the extracellular environment, the
actions of these active peptides can be terminated by proteolytic inactivation.
Therefore, chapter 10 discusses extracellular proteases involved in inactivation
of secreted peptides. However, before the extracellular proteolysis is complete,
the essential function of the released peptide is to activate its specific receptor
on the target cell to initiate certain physiological responses. Thus, chapter 11
presents the manner in which peptide hormones and neurotransmitters stimulate peptidergic receptors, with discussion of the opioid receptors as the main
example.


The authors have presented the latest developments in this field. A wealth

of knowledge has been gained over the last few years concerning the identity,
regulation, and molecular and cell biology of proteases and protease inhibitors
involved in prohormone processing. However, there are still many open areas
to investigate in this field. It is likely that there are still, as yet, unknown processing proteases to be discovered. Importantly, future knowledge of the key
proteases and regulatory components required in prohormone and
proneuropeptide processing may provide future design of clinical therapeutics
that modify the processing pathway in health and disease.


ACKNOWLEDGMENTS
I wish to thank the authors who participated in this volume for their expertise and enthusiasm in providing discussions of the current status of knowledge in the prohormone
and proneuropeptide processing field. In addition, support
from the National Institutes of Health is appreciated. Finally,
this book is dedicated to my family, who have shared with me
the excitement of science and the continuous effort that has
allowed this scientific endeavor to be achieved.



CHAPTER 1

Targeting and Activation of Peptide
Hormones in the Secretory Pathway
Ken Teter and Hsiao-Ping H. Moore

Introduction

P

rofessional secretory cells—generally cells of neuronal, endocrine, or exocrine origin—

utilize two divergent secretory pathways with distinct temporal and spatial characteristics (reviewed in refs. 1-4). The first pathway of constitutive secretion mediates the continual and unstimulated transport of lipids, membrane proteins, and soluble cargo to the
cell surface. This pathway thus provides the plasma membrane with a steady supply of protein and lipid components while it simultaneously releases secretory proteins into the extracellular space. Most cells are capable of constitutive secretion, but it was commonly thought
that the second pathway of regulated secretion was limited to professional secretory cells.
Only these cells were believed to express the specialized machinery required to sort and
store specific cargo into a subpopulation of vesicles which accumulate intracellularly until a
secretagogue triggers release. Yet recent evidence suggests that many other cell types utilize
this process as well, and the mechanism of regulated exocytosis may in fact be used for such
additional purposes as membrane repair during wound healing and the regulation of membrane permeabilities to water, ions and nutrients. As summarized in Table 1.1, regulated
exocytosis has been detected by a number of techniques in many cell types which have
previously been considered to possess only the constitutive secretory pathway. Insights gained
from the study of peptide hormone trafficking can thus be generalized to similar events
occurring in a variety of cells.
Regulated secretory vesicles can be divided into two classes based on morphology, origin and content: Dense core secretory granules (SGs) arise from the trans-Golgi network
(TGN) and contain an electron dense peptide hormone aggregate packaged in a 100-200 nm
vesicle,5-8 whereas the synaptic vesicles are electron translucent 50 nm structures which derive from the endosomal system and carry nonpeptide neurotransmitters as cargo. After
budding from the TGN, SGs are transported to the cell periphery in a microtubule-dependent process.8,9 Peptide hormone precursors are converted to a bioactive state en route by a
family of enzymes called prohormone convertases (PCs, reviewed in refs. 10-13) and are
incorporated into a highly condensed protein aggregate. Because the soluble constituents of
SGs are delivered via the secretory pathway, this route of regulated secretion is referred to as
the biosynthetic pathway. Synaptic vesicles, which recruit membrane proteins from the
endosomal system and soluble cargo from the cytoplasm, utilize what has been termed the
recycling pathway for biogenesis. The function and generation of synaptic vesicles has been
reviewed elsewhere. 4,14-17 This chapter will focus on the sorting and activation of
Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing,
edited by Vivian Y.H. Hook. ©1998 R.G. Landes Company.


stimulated release of lysosomal enzymes and pre-internalized BSA-gold complex

membrane

wound/disruption

perfusion of
cytosol with Ca2+

Ca2+ ionophore

Bovine
endothelial cells

CHO fibroblasts

NRK fibroblasts

stimulated release of sulfated GAG chains

Ca2+ ionophore

L cells;
CHO fibroblasts

229

227, 228

226

225

224

223

221, 222

Mechanisms of regulated secretion are widespread in animal cells. Although regulated secretion has historically been viewed as a specialized property of
professional secretory cells, this phenomenon has recently been characterized in other cell types and may be used for a variety of physiological processes. One
such purpose appears to be the transient modification of cell surface permeability. This facilitates the uptake of water, ions, or nutrients and is accomplished by
the stimulated translocation of membrane channels and pumps from a specialized intracellular pool to the plasma membrane. The same general mechanism
may be widely used for membrane repair during wound healing.

release of pre-loaded acetylcholine detected by patch clamp measurements of an
abutting myocyte

Ca2+ influx;
membrane
depolarization

targeting of transfected Glut4 to a unique vesicle population, presumably a storage organelle
similar to the Glut4-containing vesicle in adipocytes and muscles

vesicle accumulation and microvilli formation at wound site visualized by electron
microscopy; heightened release of pre-internalized dye in wounded cells; increase in cell size

induction of numerous exocytic ‘pores’ visualized with fluorescent dyes and confocal
microscopy; heightened release of pre-loaded dye in wounded cells

Amphibian
myocytes and
fibroblasts;

CHO fibroblasts

CHO fibroblasts; not determined
3T3 fibroblasts

increase in membrane capacitance, reflecting increased cell surface area

membrane
wound/disruption

Sea urchin eggs
and embryos;
NIH fibroblasts

220

mobilization of H+/K+-ATPase to the cell surface

histamine

Gastric cells

15, 218, 219

15, 217

References

mobilization of Glut4 glucose transporter to the cell surface

mobilization of water channels to the cell surface

vasopressin

insulin

Kidney ductules

Observation/Method of Detection

Adipocytes

Stimulus

Cell Type

Table 1.1. Nonclassical regulated secretion in animal cells

2
Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing


Targeting and Activation of Peptide Hormones in the Secretory Pathway

3

prohormones and prohormone converting enzymes along the biosynthetic regulated secretory pathway.

Trafficking and Modification of Peptide Hormone Precursors
Proteins destined for either regulated or constitutive release are transported and modified together as they migrate from the ER, through the Golgi and to the TGN. Targeting to

the regulated pathway then begins in the TGN with the preferential packaging of materials
into a budding immature secretory granule (ISG),18,19 the intermediate vesicle that is biochemically distinct from the mature SG.7,20-25 Sorting appears to continue during the period
of granule maturation, since the lysosomal enzymes and constitutively secreted proteins
that are initially incorporated into the nascent SG are found in substantially reduced quantities in the mature SG. As such, the ISG has sometimes been regarded as the functional
extension of the TGN by completing the sorting function that is initiated at that site (reviewed in ref. 26). In most cases, prohormone conversion begins in the TGN but takes place
predominantly in the ISG. This organelle therefore represents a key intermediate station for
prohormone sorting and cleavage (Table 1.2). The extent of prohormone sorting and activation occurring in the TGN vs. the ISG appears to vary for different proteins and cell types,
but in all cases the maturation process eventually generates a homogeneous SG vesicle population with highly concentrated bioactive peptides. The production of mature peptide hormones thus involves numerous sorting and processing events.

Role of ER and Golgi
Prohormone sorting and modification begins in the ER. Peptide hormones enter the
secretory pathway by virtue of a hydrophobic signal sequence which directs cotranslational
passage through a ‘translocon’ complex into the ER lumen (reviewed in refs. 27-29). Once
positioned in the ER, preprohormones are modified by a series of reactions: The signal
sequence and/or prosequence are cleaved, N-linked core carbohydrates are added, and the
prohormones are transiently associated with molecular chaperones. In the case of thyroglobulin, sequential interactions with the BiP and calnexin chaperones are required for its proper
folding.30 BiP also prevents proinsulin degradation during folding and dimerization.31 This
process, as well as proinsulin disulfide bond formation, is facilitated by the oxidizing lumenal environment of the ER.32 Since proper folding is a requisite for ER export, the time
required to complete these modifications determines, in part, the exit rate for each protein
(reviewed in ref. 33).
Until recently, secretory proteins were thought to leave the ER by default. This ‘bulk
flow’ mechanism would allow transport and constitutive secretion of proteins with no targeting information other than the initial signal sequence (reviewed in refs. 34, 35). According to this model, only residents of organelles would require specific targeting signals in
order to be retained within their respective compartments, and a variety of organelle targeting motifs have indeed been identified (Table 1.3). However, another prediction of the bulk
flow model—lack of sorting and concentration of migrant proteins upon exit from the
ER—is not supported by recent observations. Many yeast proteins, for instance, are concentrated in ER-derived carrier vesicles (reviewed in refs. 36, 37). Quantitative immunoelectron
microscopy has also established that albumin and VSV G are concentrated in ER-derived
vesicles in mammalian cells.38,39 These observations indicate that exit of migrant proteins is
facilitated by an active sorting mechanism. In the case of VSV G, this process requires a
cytoplasmic, di-acidic anterograde targeting signal.40 A phenylalanine-containing anterograde transport signal has also been found on two putative ER cargo sorting receptors, p24
and ERGIC-53.41-45 Although cognate transport signals in prohormones have yet to be

tubulo-cisternal

+++

yes

mildly acidic/neutral

yes

limited

no

no

morphology

lysosomal enzymes

clathrin coat

pH

sorting compartment

processing compartment

stimulated release

unstimulated release
yes

yes

yes

yes

5.0-7.0
average 5.7-6.3

partial

++

vesicles of irregular shape and
size, 80 nm average diameter

perinuclear to peripheral

ISG

limited

yes

limited

no

5.0-5.5

no

+/–

100-200 nm
vesicles

peripheral

SG

23, 82, 118, 153, 155

8, 118, 148, 153, 155

20, 82, 107, 108, 121, 153,
174, 178-180, 182, 183,
188, 200, 201, 230-234

18, 19, 24, 118, 145, 146,
149, 154, 157, 158

9, 108, 188, 200-202,
204-206

7, 20-22, 24

149, 158, 159

6, 8, 22

22, 24, 118

References

Serving as an intermediate between the TGN and SG, the ISG shares characteristics of both organelles. Like the TGN, the ISG is decorated with γ-adaptin and clathrin,
contains lysosomal enzymes, maintains an average pH of approximately 6.2, and acts as sorting station for routing secretory traffic to multiple sites. Prohormone
processing also takes place in both the TGN and ISG, although in most cases cleavage in the TGN is fairly limited. Another major difference between the two organelles
is the response to secretagogues: Only the ISG is capable of stimulated exocytosis. The ISG resembles the mature SG in this respect, although stimulation usually
results in the preferential exocytosis of young granules. In addition, the contents of newly formed ISGs exhibit a higher rate of unstimulated release. In some cases,
this secretion has been shown to result from the budding of transport vesicles from ISGs. With time, however, the maturation process places release from ISGs under
tight regulation and eventually transforms the vesicles into a uniform population of SGs. Note that the characteristics summarized in the table above are derived from
numerous studies involving different cell types, marker proteins and model systems. This variability may account for some of the inconsistencies present in the
literature.

perinuclear

location

TGN

Table 1.2. Relationship between the TGN, ISG and SG

4
Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

Targeting and Activation of Peptide Hormones in the Secretory Pathway

5

identified, trafficking of these proteins may also involve ER export signals and cargo sorting
receptors.
By contrast, bulk flow may apply to intra-Golgi transport since further concentration
of migrant proteins does not occur as trafficking continues across the Golgi stacks.38,39,46
While the exact mechanism of intra-Golgi transport is currently under debate,47-49 it is clear
that a number of protein modifications occur at this site. Proinsulin hexamerization is initiated in an early Golgi compartment by the addition of zinc,32,50 and a subset of regulated
secretory proteins are phosphorylated in the trans-Golgi.51 A prevalent modification is the
addition of N- and O-linked carbohydrate chains, generated by the coordinate action of a
characteristic set of enzymes in each Golgi cisternae (reviewed in refs. 52,53). The multiple
Golgi cisternae also appear to act as a “molecular sieve”54 which allows repeated opportunities for the capture and return of missorted ER resident proteins. This retrieval process is
mediated by a set of carrier vesicles bearing the coat complex COPI (reviewed in
refs. 36,55,56). Proteins destined for secretion appear to be excluded from these retrograde
transport carriers, as recent studies indicate that proinsulin and VSV G protein are segregated into distinct vesicles from those containing the ER retrieval KDEL receptor.46 Interestingly, the proinsulin-containing vesicles are also COPI-coated. The possibility that COPI
functions in both anterograde and retrograde transport remains an issue that requires further clarification.56-59

Role of TGN, ISG and SG
The TGN serves as a major sorting station for secretory traffic, diverting proteins to
regulated, constitutive, lysosomal, and (in polarized cells) apical or basolateral destinations
(reviewed in refs. 60, 61). Recent studies indicate that in nonpolarized mammalian cells and
in yeast, different classes of constitutive vesicles, each with a distinct set of cargo, are also
generated by the TGN.62-64 Its central role in these trafficking events has made the TGN a
subject of numerous studies. Originally defined as the site at which newly synthesized plasma
membrane proteins accumulate at 20°C, the TGN could be visualized by electron microscopy as a tubulocisternal network directly apposed to the trans-Golgi cisternae.65-67 Later, it
was defined by two biochemical reactions—sialation and tyrosine sulfation—which occur

at this site.68,69 The intracellular distributions of TGN38, furin and the mannose 6-phosphate receptor have also been used to delineate the compartment.70-74 Thus, over time the
TGN has been defined by many different parameters and markers. These definitions have
been used interchangeably, but a close examination of the literature reveals considerable
inconsistencies regarding the response of the TGN to drug treatment (Table 1.4). This raises
the possibility that the organelle known as the “TGN” may in fact be referring to more than
one compartment. Because models for prohormone sorting and activation rely critically on
the exact locations in which these events occur, the definition of the TGN needs to be revisited in order to avoid confusion caused by inconsistent usage of the term “TGN”.
In addition to sulfation and sialation, one other major prohormone modification—
processing to a bioactive state—is initiated in the TGN. Functional peptide hormones are
usually quite small but are often synthesized as larger, inactive precursors which are eventually cleaved by the PC enzymes. Each prohormone convertase recognizes different dibasic
consensus sequences, so the generation of a specific bioactive peptide relies upon the activity of one or more PCs. As a result, the same prohormone can yield different products
depending on which PC(s) it encounters.75-78 Prohormone cleavage also exposes C-terminal basic amino acids which are recognized and removed by another modifying enzyme,
carboxypeptidase E (CPE, reviewed in ref. 79). In some instances, alpha-amidation may
also follow prohormone cleavage (reviewed in ref. 80).


241
241
144, 242, 243
244
242, 243

intercalates into glycolipid ‘rafts’ destined for apical membrane
interaction with the lectin-like, carbohydrate binding receptor VIP36
unknown mechanism

238
166, 167, 239
166, 167, 240

unknown retention mechanism;may involve “kin recognition” or an
interaction with the Golgi lipid bilayer
unknown retention mechanism
unknown retention mechanism;involves phosophorylation state
retrieval mechanism most likely involving cytoplasmic coat
(clathrin and AP-2) proteins
addition of phosphate to a mannose residue in the cis Golgi
pH-dependent sorting mechanism involving a receptor in the TGN

55, 237

236

235

36, 55, 56

36, 55

References

Protein targeting requires specific localization signals. Organelle residence is established by a combination of retention and retrieval. The retention mechanism
prevents most resident proteins from continuing along the secretory pathway, while an auxillary retrieval mechanism captures errant proteins in distal secretory
compartments and returns them to the proper organelle. Targeting is most often due to either a unique physical property of the protein or to a receptor/motif interaction.
Variations on these two themes are used throughout the secretory pathway, including diversion to the SG, lysosome, or polarized plasma membrane.

transmembrane domain (TGN38)
cytoplasmic acidic cluster (furin)
cytoplasmic tyrosine tight-turn
motif (TGN38 and furin)

Lysosomes
specific conformational motif
mannose 6-phosphate residue
Epithelial plasma membrane
apical surface
GPI lipid anchor,
transmembrane domain
N-linked glycans
basolateral surface
cytoplasmic tyrosine tight turn
motif or dileucine sequence

TGN

transmembrane domain

unknown retrieval mechanism

cytoplasmic
N-terminal RR motif

Golgi

unknown retention mechanism

transmembrane domain

type II membrane
proteins

retrieval mechanism effected by cytoplasmic coat (COPI) proteins

pH-dependent retrieval mechanism involving a KDEL receptor

TargetingMechanism

cytoplasmic
C-terminal KK motif

lumenal
C-terminal KDEL tag

Targeting
Determinant

type I membrane
proteins

ER
soluble proteins

Destination

Table 1.3. Targeting signals for organelles along the secretory path

6
Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing


large vesicular structures

ER
ER
cofractionates with TGN38
ER
not determined
MTOC
MTOC
MTOC

20° block site
VSV G viral protein
SFV viral proteins
secreted GFP fusion construct
radiolabeled proinsulin
Sialyltransferase compartment
Site for sulfation
TGN38 compartment
Furin compartment
MPR compartment
blocked
blocked
blocked
blocked
not applicable
blocked
enhanced
not inhibited
not inhibited

Export in the Presence of BfA

245
246
247
248
249-252
82, 148, 182, 245, 253
254, 255
73, 256
257

References

Serving as a major sorting station for secretory traffic, the TGN has been the subject of numerous studies and can be defined by a number of parameters. Yet a survey
of the literature reveals that these parameters produce conflicting results when subjected to Brefeldin A (BfA) treatment. In the presence of BfA, a fungal metabolite
which induces the redistribution of Golgi residents to the ER,258 the TGN (as defined by TGN38, furin, or MPR) did not collapse to the ER but was instead found at
a tubularized perinuclear site which also contained internalized transferrin. This established the TGN as a functionally and physically distinct organelle, separate
from the trans-Golgi. However, the TGN does redistribute to the ER when it is defined by other criteria (i.e., the 20° block site or the sialyltransferase compartment).
This inconsistency and the differential effect of BfA on TGN transport leave open the possibility that two distinct compartments are both being defined as the TGN.
Further examination of the nature of the TGN and the relationship between the various definitions of the compartment may thus help to clarify some of the
discrepancies regarding the site of prohormone sorting and activation.

Location in the Presence of BfA

Definition

Table 1.4. Definitions of the TGN and responses to Brefeldin A

Targeting and Activation of Peptide Hormones in the Secretory Pathway
7

8

Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

Prohormone cleavage and activation continues in the ISG, the vesicular intermediate
linking the TGN and SG. Concomitant with this event are other steps of granule maturation: Translocation to the cell periphery, progressive lumenal acidification, homotypic fusion between two or more ISGs, and loss of the clathrin coat with the simultaneous removal
of missorted proteins and excess membrane (reviewed in refs. 25,26,81). Many of the
missorted proteins are lysosomal and constitutive secretory proteins, but membrane proteins of the constitutive fusion machinery may also be incorporated into the budding ISG.
Mistargeting of components of this fusion machinery could explain the high level of
unstimulated release of ISGs early in the maturation process.82 In contrast, other proteins
required for regulated exocytosis are apparently added to the ISG. The SG membrane protein VAMP-2, for example, is delivered to the nascent SG from a post-Golgi site (B. Eaton
and H.-P. Moore, manuscript in preparation). The delayed delivery of components of the
regulated fusion machinery could also account for the short lag period in which newly formed
ISGs are refractory to stimulated release in pituitary AtT20 cells (M. Haugwitz and H.-P.
Moore, manuscript in preparation). SG biogenesis is thus a multi-step process involving
numerous budding and fusion events which serve to alter both the soluble and membrane
composition of an ISG.

Prohormone Sorting Mechanisms
The mechanisms for targeting peptides to the regulated secretory pathway appear to
have been conserved within the family of professional secretory cells: Regulated proteins
which are not endogenously expressed by a particular professional secretory cell are still
recognized and stored in SGs when introduced into that cell by DNA transfection.83 This
indicates that the regulated sorting machinery recognizes common determinants present in
many secretory proteins and explains why multiple secretory products can often be found
within the same granule.18,84-88
As with other intracellular targeting events, there are two conceptually distinct mechanisms that may explain sorting to the regulated pathway. Sorting may be accomplished by a
receptor which recognizes a targeting motif on specified proteins and in turn delivers them

to the proper compartment. Typically, the receptor would cycle between the sorting and
target compartments to mediate multiple rounds of binding and dissociation. An alternative mechanism involves the formation of specific macromolecular complexes between
molecules destined for the same organelle. This would entail a conformational transition
that initiates the formation of these molecular aggregates at the site of sorting. This mechanism thus allows multiple components to be sorted synchronously without the need of a
cycling receptor. Numerous studies in recent years have uncovered specific interactions between various protein components of the regulated secretory pathway. To date, a cycling
receptor that mediates multiple rounds of binding and dissociation has not been found.
Instead, the available data support a model in which the formation of specific macromolecular aggregates of granule components dictates their coordinate sorting into regulated
secretory granules. These interactions can be divided into two types: content-to-content
interactions, and content-to-membrane interactions.

Interactions between SG Contents
Homotypic interactions
Proteins targeted to the regulated pathway are highly concentrated in the mature SG
(reviewed in refs. 1, 2, 89). Electron microscopic studies have shown that the concentration
process begins at the dilated rim of the trans-Golgi (reviewed in refs. 89, 180), thus leading
to the hypothesis that selective aggregation of soluble SG content plays a key role in sorting