signal transduction: pathways, mechanisms and diseases · signal transduction: pathways, mechanisms...

Signal Transduction: Pathways, Mechanismsand Diseases

Ari SitaramayyaEditor

Signal Transduction:Pathways, Mechanismsand Diseases

EditorAri SitaramayyaProfessor of Biomedical Sciences423 DHEEye Research InstituteOakland UniversityRochester, MI [email protected]

ISBN 978-3-642-02111-4 e-ISBN 978-3-642-02112-1DOI 10.1007/978-3-642-02112-1Springer Heidelberg Dordrecht London New York

Library of Congress Control Number: 2009928304

# Springer-Verlag Berlin Heidelberg 2010This work is subject to copyright. All rights are reserved, whether the whole or part of the material isconcerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publicationor parts thereof is permitted only under the provisions of the German Copyright Law of September 9,1965, in its current version, and permission for use must always be obtained from Springer. Violations areliable to prosecution under the German Copyright Law.The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply,even in the absence of a specific statement, that such names are exempt from the relevant protective lawsand regulations and therefore free for general use.

Cover illustration: Cellular and subcellular detection of M 2 receptors in striatal neurons; see Fig. 2.1 inChap. 2 ‘‘Regulation of Intraneuronal Trafficking of G-Protein-Coupled Receptors by NeurotransmittersIn Vivo’’ by Veronique Bernard

Cover design: WMXDesign GmbH, Heidelberg, Germany

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

For Usha, Vani, and Aruna

Preface

Teaching a graduate course on signal transduction in the fall of 2007 was very

enjoyable. Each week, my students and I discussed a recently published research

paper in some area of signaling, argued about the appropriateness of the methodol-

ogy used in the research, the design of the experiments, and whether the conclu-

sions drawn were supported by the results presented. Finally, each student was

asked to list what was good and what was deficient in the paper and whether she/he

would have accepted or rejected it had she/he reviewed it for publication. For a

good number of recent papers, a consistent complaint of the students was that the

background to the research was not adequately described. It can of course be argued

that students should research the background. Either way, the need for a good

source of background material in order to appreciate the research presented in a

paper became apparent to me and was the inspiration for developing this book.

The goal in bringing this book was to provide students with a review of recent

developments in specific areas of current interest in signal transduction, sufficiently

in depth to make recent research publications accessible. However, given the wide

range of research topics being investigated today in signaling, a choice had to be

made to focus on a select few. This choice was made not only keeping the current

research interest in mind, but also with the anticipation that these areas will

continue to be of interest over the next several years.

In choosing papers for discussion in my class, I look for health-relatedness of the

research, and that is reflected in the list of the broad areas covered in this book – G

protein coupled receptors, growth factors, nuclear receptors, reactive oxygen and

nitrogen species, cell cycle and cancer; the emphasis has been on areas of signaling

research with immediate clinical significance. I believe this book will serve well as

supplemental reading material for undergraduate and graduate students with similar

interest.

Another area covered in this book, one not often highlighted in signal transduc-

tion books, is that of signaling platforms, which is emerging as a significant area of

research relevant to cellular metabolism, cell proliferation, differentiation, apopto-

sis, neurodegenerative diseases, and cancer. The authors contributing to this book

vii

are experts and active contributors to research in their specific areas of signaling.

I would like to thank them for their contributions and their suggestions in the

development of the book. I would like to acknowledge the help of Frank Tedeschi,

a student in my department, who assisted in editing the manuscripts and in prepar-

ing the index. I would also like to thank Dr. Sabine Schwarz, Life Sciences Editor at

Springer Verlag, and Dr. Jutta Lindenborn who have been very helpful at every

stage of production of this book.

Ari Sitaramayya

viii Preface

Contents

Part I G Proteins and G Protein–Coupled Receptors

1 Traditional GPCR Pharmacology and Beyond . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Annette Gilchrist and Maria R. Mazzoni

2 Regulation of Intraneuronal Trafficking of G-Protein-Coupled

Receptors by Neurotransmitters In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Veronique Bernard

3 Small GTPases and Their Role in Regulating G Protein-Coupled

Receptor Signal Transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Fabiola M. Ribeiro and Stephen S.G. Ferguson

4 Regulation of G Protein Receptor Coupling, Mood Disorders

and Mechanism of Action of Antidepressants . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Moran Golan, Gabriel Schreiber, and Sofia Avissar

5 Dysregulation of G Protein-Coupled Receptor Signaling

in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

JoAnn Trejo

Part II Growth Factors

6 Insulin Signaling in Normal and Diabetic Conditions . . . . . . . . . . . . . . . . . 101

Patrice E Fort, Hisanori Imai, Raju Rajala, and Thomas W. Gardner

7 Epidermal Growth Factor (EGF) Receptor Signaling

and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Elizabeth S. Henson and Spencer B. Gibson

ix

8 Leptin Signaling Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

Hiroyuki Shimizu

9 Signaling in Normal and Pathological Angiogenesis . . . . . . . . . . . . . . . . . . 159

Michael R. Mancuso and Calvin J. Kuo

Part III Signaling Platforms

10 Spatial and Temporal Control of Cell Signaling

by A-Kinase Anchoring Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

F. Donelson Smith, Lorene K. Langeberg, and John D. Scott

11 Mitochondria, a Platform for Diverse Signaling Pathways . . . . . . . . . . 199

Astrid C. Schauss and Heidi M. McBridee

12 Mitogen-Activated Protein Kinases

and Their Scaffolding Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

Danny N. Dhanasekaran and E. Premkumar Reddy

13 Molecular and Functional Determinants of Ca2þ

Signaling Microdomains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237

Indu S. Ambudkar, Hwei L. Ong, and Brij B. Singh

Part IV Nuclear Receptors / Transcription

14 Eukaryotic Gene Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

Jennifer H. Gromek and Arik Dvir

15 Estrogen Signaling Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273

Dapeng Zhang and Vance L. Trudeau

16 Signal Transduction Pathways Involved

in Glucocorticoid Actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289

Peter J. Barnes

Part V Reactive Signaling Molecules

17 Cellular Signaling by Reactive Oxygen Species: Biochemical

Basis and Physiological Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313

Michel B. Toledano, Simon Fourquet, and Benoıt D’Autreaux

18 Soluble Guanylyl Cyclase: The Nitric Oxide Receptor . . . . . . . . . . . . . . . 337

Doris Koesling and Ari Sitaramayya

x Contents

Part VI Cell Cycle, Cell Death and Cancer

19 Distinct Roles of the Pocket Proteins in the Control

of Cell Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353

Paraskevi Vogiatzi and Pier Paolo Claudio

20 Activation of the p53 Tumor Suppressor and its Multiple Roles

in Cell Cycle and Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375

Luciana E. Giono and James J. Manfredi

21 Aging and Cancer: Caretakers and Gatekeepers . . . . . . . . . . . . . . . . . . . . . 397

Diana van Heemst

22 Signal Transduction in Embryonic Stem Cells

and the Rise of iPS Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417

Solene Jamet, Ruairi Friel, and P. Joseph Mee

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427

Contents xi

Part IG Proteins and G Protein–Coupled

Receptors

3A. Sitaramayya (ed.), Signal Transduction: Pathways, Mechanisms and Diseases, DOI 10.1007/978-3-642-02112-1_1, © Springer-Verlag Berlin Heidelberg 2010

Chapter 1Traditional GPCR Pharmacology and Beyond

Annette Gilchrist and Maria R. Mazzoni

A. Gilchrist (*) Midwestern University, 555 31st Street, Downers Grove, IL 40515, USAe-mail: [email protected]

M.R. Mazzoni University of Pisa, Via Banano 6, Pisa 56126, Italy

Abbreviations

AGS Activator of G protein signalingAR Adrenergic receptorCRE cAMP response elementDAG 1,2 diacylglycerolERK Extracellular signal related kinaseFRET Fluorescence resonance energy transferG proteins Guanine nucleotide binding proteinsGEF Guanine exchange factorGIP G protein coupled receptor interacting proteinGLP Glucagon-like peptideGPCR G protein coupled receptorGRK G protein coupled receptor kinaseHTS High-throughput screeningIP

3 Inositol 1,4,5-trisphosphate

PIP2 Phosphatidylinositol 4,5-bisphosphate

RGS Regulator of G protein signalingTM Transmembrane

1.1 Introduction

Many biologically active molecules convey their signals via G protein coupled receptors (GPCRs) which are coupled to heterotrimeric guanine nucleotide binding proteins (G proteins). GPCRs respond to a large variety of molecules from small

4 A. Gilchrist and M.R. Mazzoni

molecules such as acetylcholine, norepinepherine, serotonin, dopamine, and hista-mine to large peptides and glycoprotein hormones. There are over 800 GPCRs in the human genome, and the expansive list has resulted in GPCRs frequently being the focus for those searching for new therapeutics. But GPCRs are not unique to mam-mals as seven transmembrane (7TM)-containing proteins have now been identified in five of the six kingdoms of life (Bacteria, Protozoa, Plantae, Fungi and Animalia).

Genetic variation in GPCRs, leading to inactive, overactive, or constitutively active receptors, has been shown to be present in a wide variety of human diseases (Thompson et al. 2008a). Interventions aimed at correcting genetic GPCR dysfunc-tion include calcimimetics used to compensate for some calcium sensing receptor mutations, compounds that revert the gonadotropin releasing hormone receptor loss from the cell surface which is found in idiopathic hypogonadotropic hypogonad-ism, and novel drugs to rescue the purinergic P2Y

12 receptor which results in a rare

bleeding disorder.By excluding the sensory receptors such as the olfactory, bitter taste, vomerona-

sal, sweet/umami taste and opsin/rhodopin related receptors, it has been suggested that, of the over 800 GPCRs, some 369 are “physiologically relevant” and could serve as drug targets (Lagerström and Schiöth 2008). Of these, the activating ligand has not been identified for approximately 150 of these receptors, and they remain “orphans.” A continually updated list of GPCRs is available online at http://www.iupharbb.org/receptorList/results.php (Foord et al. 2005).

Members of the GPCR superfamily are diverse in their primary structure, and this has been used for the phylogenetic classification of the family members such as that provided by Kolakowski in 1994 with an A–F classification system (Kolakowski 1994). A similar but extended nomenclature system was introduced in 1999, with the receptors being divided into five families on the basis of structural and ligand-binding criteria (Bockaert and Pin 1999). Using a draft of the human genome which became available in 2001, Fredriksson and colleagues divided 802 (known and predicted) human GPCRs into five families on the basis of phylogenetic criteria, termed Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2 and Secretin (GRAFS) (Fredriksson et al. 2003). One can also use BIAS-PROFS (http://igrid-ext.cryst.bbk.ac.uk/gpcrtree/), an internet site that classifies GPCRs at the class, subfamily and sub-subfamily level when a protein sequence is entered (Davies et al. 2008)

Although for years the underlying premise of GPCR signaling centered on their ability to couple to G proteins, this fundamental paradigm of receptor biology has been expanded as research showing GPCRs participating in protein–protein interactions independent of G proteins has emerged. Some of the GPCR–protein interactions appear to serve as scaffolds, to tether the receptor to particular subcel-lular locations or to effector enzymes and regulatory proteins. Several of these proteins control receptor trafficking, while others modulate the kinetics of GPCR-mediated signaling transduction (Wang and Limbird 2007). Many GPCR-associated proteins bind other signaling molecules suggesting that they serve to provide a means to signal via non-G protein-regulated effectors (Bockaert et al. 2004). In fact, several GPCRs have been shown to couple (directly or through adapter pro-teins) to enzymatic effectors, including guanine exchange factors (GEFs) for small G proteins, non-receptor tyrosine kinases, and components of the extracellular

51 Traditional GPCR Pharmacology and Beyond

signal related kinase (ERK) pathway, indicating that GPCR signal transduction can occur independent of heterotrimeric G proteins. An example of GPCR/G protein-independent signaling is through b-arrestins which bind to activated, phosphory-lated receptors and control internalization of the receptors, G protein coupling, and secondary signaling that is independent of G protein signaling. The events appear to be differentially regulated by GPCR kinase (GRK) phosphorylation, ubiquitina-tion and b-arrestin oligomerization, which may be both receptor- and cell-type-dependent (Dromey and Pfleger 2008).

There is a bias to the GPCRs that have been successfully targeted, with com-pounds being identified for > 39 Rhodopsin (class A) receptors, four Secretin (class B) receptors, and three Glutamate (class C) receptors. Of those GPCRs that have proved to be good therapeutic targets, 17% (23/133) bind peptides or proteins (including enzymes); 29% (12/41) bind biogenic amines; 20% (7/35) bind lipid-like-molecules; 17% (2/12) bind amino acids, and 6% (1/16) bind purines. Perhaps most surprising is that even with such a large number of candidates, only 46 GPCRs (~12%) have been successfully targeted by drugs (Lagerström and Schiöth 2008). Yet, nearly half of all prescription drugs were targeted towards GPCRs in 2000 in the United States (Drews 2000). No doubt this success provides the financial basis for the ongoing pursuit by pharmaceutical companies in exploring both new and old targets from this important receptor family.

The common structural feature of GPCRs is 7TM a-helical domains, each com-posed of 25–35 amino acid residues. The highly hydrophobic helices are connected by three extracellular and three intracellular loops, with the amino (N-) terminus being extracellular and the carboxyl (C-) terminus being intracellular (Fig. 1.1). This structure gave rise to an alternative name for these molecules, 7TM receptors. One of the greatest advances in GPCR knowledge came with the high-resolution crystal structure of inactive rhodopsin (Palczewski et al. 2000), which was eventu-ally followed with the crystal structures for rhodopsin with a deprotonated reti-nylidene Schiff base linkage (Salom et al. 2006), b

2-adrenergic receptor (AR)

(Cherezov et al. 2007; Rasmussen et al. 2007), b1-AR (Warne et al. 2008), squid

rhodopsin (Murakami and Kouyama 2008; Shimamura et al. 2008), and active rhodopsin (Park et al. 2008). Moreover, the surface movement of rhodopsin upon photoactivation was mapped (Altenbach et al. 2008) using an electron pair spin resonance technology called “double electron–electron resonance” spectroscopy, which interrogates pairs of nitroxide spin labels to provides a solid foundation for the so-called “helix movement model” of receptor activation.

Stimulation of heterotrimeric G proteins via GPCRs results in the regulation of a variety of intracellular signaling pathways through activation of downstream effectors such as enzymes, ion channels, and nuclear transcription factors. Heterotrimeric G proteins are comprised of a Ga subunit that binds and hydrolyzes GTP, and a Gbg dimer that serves as a functional monomer. The Gabg holoprotein acts as a molecular switch that can be turned “on” and “off” via the GTPase cycle (Fig. 1.2). Numerous members comprise the heterotrimer G protein family, includ-ing genes for 16 Ga subunits, five Gb subunits, and 14 Gg subunits (Milligan and Kostenis 2006). The Ga subunits are a family of 39–52 kDa proteins commonly divided into four families based on their sequence similarity: G

i/o, G

s, G

q/11, and


G12/13

. Several of the Ga subunits are expressed ubiquitously, while others have temporal and/or differential expression (Eglen et al. 2008). Intensive studies of Ga proteins have helped elucidate distinct regions involved in receptor recognition, GTP binding and hydrolysis, guanine nucleotide-induced conformational changes, and effector interaction. Structurally, each Ga subunit consists of two domains, a GTPase domain and an a helical domain. In between these two domains is a cleft where the guanine nucleotide binds (Tanabe et al. 1985). Lipid modification of a Cys residue near the N-terminus of most Ga subunits allows for binding to mem-brane (Linder et al. 1993) while the C-terminus of all Ga subunits appears to be important for interaction with the receptor. Gbg is an absolute requirement for the binding of the Ga subunit to the receptor, for the formation of high-affinity agonist binding, and for receptor catalyzed activation of G protein (Fung 1983; Blumer and

I II

IV

III

V

VIVII

extracellular space

cytosol

EC1

EC2

EC3

IC1

N-

C-

P

PP

PIC2

IC3

Ag

αβ

γ

HeterotrimericG-protein

I II

IV

III

V

IC1

P

PP

P

I II

IV

IIIV

IC1

P

PP

P

Ag

αβ

γ

Fig. 1.1 Topology of a typical GPCR. The receptor’s N-terminus is extracellular, and its C-terminus is intracellular. The receptor traverses the plane of the cell membrane seven times. The hydrophobic transmembrane segments (light color) are designated by roman numerals (I–VII). The agonist (Ag) approaches the receptor from the extracellular side, while G proteins interact with cytoplasmic regions of the receptor, especially portions of the third cytoplasmic loop between transmembrane regions V and VI. The receptor’s cytoplasmic terminal tail contains numerous serine and threonine residues whose hydroxyl (–OH) groups can be phosphorylated, and this phosphorylation is often associated with receptor internalization


Thorner 1990). Gbg is also tethered to the membrane by a lipid modification of the Gg subunit and together the Gbg dimer acts as a scaffolding protein. Following receptor activation, both Ga and Gbg act as signaling molecules (Fig. 1.2).

Although much has been learned about how ligand-stimulated GPCRs medi-ate G protein activation, the exact mechanisms along the way, such as the exis-tence of precoupled G proteins, the role of GEFs in nucleotide exchange on Ga, and dissociation of Ga and Gbg from the receptor, are now being considered in greater detail. A number of studies have shown that G proteins may not physically dissociate from the receptors (Frank et al. 2005; Digby et al. 2006; Yuan et al. 2007). In addition, there appear to be several downstream effectors that can be activated by the GPCR independent of nucleotide exchange (Sun et al. 2007). There has also been recognition that other proteins such as GRKs, activator of G protein signaling (AGS) proteins, regulator of G protein signaling (RGS) proteins,

GDP

GDP

GTP

Piα

βγ

RGS

+

Downstream Effectors

αβγ

GTPα β

γ

IIIIIII V

VIVII

I

IVVIIIIIIII

VI

extracellular space

cytosol

InactiveG-protein

‘7TM’ - receptor

Ligand

GDP-GTPExchange

ActiveGα

ActiveGβγ

I VIV

VII

VIIIIII

VII

Fig. 1.2 Activation of G protein signaling via ligand binding to a GPCR. Following ligand-initi-ated stimulation, the 7TM receptor becomes activated and changes its conformation leading to an interaction between Gabg and the GPCR. This interaction induces a conformation change in Ga that decreases its affinity for GDP whereby it dissociates and is replaced with GTP. Once GTP is bound, the Ga subunit assumes an activated conformation and rearrange/dissociates from receptor and Gbg. Both Ga-GTP and Gbg initiate downstream signaling events. The activated state lasts until the GTP is hydrolyzed to GDP by the intrinsic GTPase activity of the Ga subunit, resulting in association of the Ga-GDP with Gbg subunits to form inactive Gabg, and this process may be influenced by RGS proteins


and GPCR interacting proteins (GIPs) may play an important role in the activation and deactivation processes (Bockaert et al. 2004; Jean-Baptiste et al. 2006; Blumer et al. 2007; Ribas et al. 2007). Adding to the complexity is the substantial evidence indicating that posttranscriptional modifications, receptor oligomerization, and the presence (or absence) of specific G proteins can influence GPCR activation and signaling (Gilchrist 2007).

Activation of a GPCR results in the release of GDP from Ga. Until GTP binds, a high-affinity complex is formed between the receptor and “empty pocket” G protein. Current structural models of the receptor with the G protein suggest that the nucle-otide-binding pocket of Ga is at a distance from the closest receptor contact site (~30 Å) lending to the “action at a distance” hypothesis (Oldham and Hamm 2008). The exchange of GDP for GTP leads to dissociation of the Gbg dimer from the Ga subunit, and both initiate unique intracellular signaling responses often referred to as second messengers (Sprang et al. 2007). The second messengers go on to activate or inhibit other components of the cellular machinery. For example, activation of G

q

may lead to activation of phospholipase C, an enzyme that catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP

2) to 1,2-diacylglycerol (DAG) and

inositol 1,4,5-trisphosphate (IP3). IP

3 interacts with receptors on intracellular cal-

cium stores resulting in cytosolic release of calcium while DAG can activate protein kinase C isoforms. Other G proteins modulate a large number of second messenger systems including cyclic AMP, cyclic GMP, calmodulin, and kinases.

Attempts to understand how a receptor catalyzes G protein activation has led to the proposition of several models including the “lever-arm,” “gear-shift,” and “latch” models as mechanisms by which the GDP is released (Johnston and Siderovski 2007). In such models, the interactions between Ga and Gbg have a crucial role in G protein activation. With the lever-arm model, the GPCR uses the N-terminal helix of Ga as a lever arm to pull Gbg away from Ga, which then forces open the nucleotide-binding pocket resulting in GDP release (Rondard et al. 2001). In the gear-shift model the GPCR uses the aN-helix to force Gbg into Ga, permit-ting the Ga N-terminus to engage the helical domain, resulting in an interdomain gap between the helical and GTPase domains of the Ga, thus leading to GDP release (Cherfils and Chabre 2003). In the latch model (Nanoff et al. 2006) it is proposed that the GPCR can engage the C-terminus of Ga to destabilize nucleotide binding from the “back side” of the nucleotide binding pocket.

Although the studies emphasize the uncertainty that still surrounds the exact mechanisms of GPCR–G protein activation, structural studies have provided detailed information on the three-dimensional structures (Wall et al. 1995; Lambright et al. 1996; Sondek et al. 1996), including localization of the nucleotide-binding pocket of Ga which is approximately 30 Å away from the nearest recep-tor contact site. Thus, the activated receptor must induce conformational changes such that upon interaction with the G protein the signal is transmitted over this distance resulting in GDP release (Ciarkowski et al. 2005). Recently, site-directed spin-labeling studies have shown that several regions in the Ga subunit undergo changes in mobility upon receptor binding (Oldham et al. 2006). Besides several C-terminal residues, other distant residues show changes in mobility consistent with


rotation/translation of the a5 helix towards the b6 strand and rearrangement of switch I and switch II regions at the Gb-binding surface. Moreover, the a5 helix is in close proximity to the receptor-binding C-terminus of Ga and the guanine-ring-binding TCAT motif is in the b6–a5 loop connecting the b6 strand to the a5 helix. In order for GDP to be released, some of the contacts between the GTPase and the helical domains of Ga may be broken. Although experimental data do not support a role of interdomain interactions in G protein activation, inspection of the crystal structure seems to suggest the requirement of a movement of these two domains relative to each other to release GDP. Indeed, both molecular dynamic simula-tion (Ceruso et al. 2004) and site-directed mutagenesis in the interdomain linkers (Majumdar et al. 2004) suggest an interdomain reorientation.

The G protein heterotrimer is reformed by GTPase activity of the Ga subunit, forming Ga-GDP and by doing so, allowing Ga and Gbg to reassociate/recombine (Fig. 1.2). This terminates the action of the Ga and Gbg subunits on their effectors. Agonist activation increases the rate of guanine nucleotide exchange and, therefore, the amount of active Ga-GTP and free Gbg. RGS proteins increase the rate of hydrolysis of GTP and thus shorten the lifetime of Ga-GTP and therefore of free Gbg. Consequently, the G proteins regulate both the specificity and duration of the initiating signal. In recent years, additional methods of regulating signaling through heterotrimeric G proteins have been identified, thus widening the perspective on the role of a G protein as a “signaling switch.” Besides RGS proteins, other accessory proteins can interact with G proteins and some of these interactions may be inde-pendent of receptor activation. In this regard, receptor-independent AGS proteins have emerged (Cismowski 2006). These proteins are classified into different groups depending on their role in signal transduction. Some AGS proteins such as AGS1 increase GTPgS binding to both Ga

i subunits and heterotrimeric G

i proteins acting

as putative GEFs (Cismowski et al. 2000), while others such as AGS8 bind to Gbg and activate it in the heterotrimeric complex without causing subunit dissociation (Yuan et al. 2007). Indeed, some experimental evidence indicates that subunit dis-sociation is not necessarily a required step in the G protein activation cycle. A fluo-rescence resonance energy transfer (FRET) study has shown that G

i1 protein

activation in cells involves subunit rearrangement rather than dissociation (Bünemann et al. 2003). Other investigations using fluorescence recovery after photobleaching have shown that some G protein subunits (G

i3 and G

oA) physically

dissociate in living cells while other heterotrimers (Gs) do not (Digby et al. 2006).

It seems from the experimental work thus far that more extensive studies will be required to obtain a complete knowledge of G protein activation and signaling in living cells.

Some GPCRs may assume active conformations in the absence of agonists and thus, constitutively or spontaneously, activate G proteins. An example is the hista-mine H

3 receptor which shows a high level of constitutive activity both in vitro and

in vivo. Constitutive activity has been observed for many other wild-type GPCRs from humans and commonly used laboratory animals. However, there are docu-mented differences in the extent of constitutive activity among GPCRs, even between subtypes of the same receptor family (Milligan 2003). Even for those


receptors that have little or no constitutive activity, such as rhodopsin, constitutively activating mutations (CAMs) can increase the level of spontaneous activity (Parnot et al. 2002). There are several indications that constitutive activity may reflect the inherent flexibility of a GPCR and its tendency to exist in more than one conforma-tion in the absence of ligands (Kobilka and Deupi 2007). For a variety of GPCRs, point mutations have been clearly linked to human disease. For example, there are approximately 50 different CAMs found in the thyroid-stimulating hormone recep-tor which are associated with hyperfunctioning thyroid adenomas. Additionally, GPCRs encoded and expressed in infected cells by herpesviruses such as Epstein–Barr virus, human cytomegalovirus, and Kaposi’s sarcoma-associated herpesvirus (KSHV) are interesting examples of the pathophysiological importance of constitu-tive GPCR activity. The GPCR encoded by KSHV acts as an oncogene and angio-genesis activator, suggesting a role of somatic CAMs in cancer development. In view of the potential role of herpesvirus-encoded GPCRs in viral dissemination and redirection of intracellular signaling pathways, they are exploited as targets for drug discovery efforts to identify compounds that can inhibit their constitutive signaling (inverse agonists).

There are a number of regions on G protein a subunits that could serve to block receptor–G protein interactions. For example, with all G proteins studied the GTP- and Mg2+-binding sites are tightly coupled. Dominant-negative constructs of the Ga subunit have been made in which mutations are introduced at residues known to contact the magnesium ion. For the Ga subunit, this includes mutations of the Gly residue within the invariant sequence (G203T; G204A) (Hermouet et al. 1991; Murray-Whelan et al. 1995), as well as mutations of a Ser residue in the effector loop, switch I region (S47C) (Slepak et al. 1993). Although this approach was quite successful with p21ras and other small G proteins, dominant-negative Ga subunits using this method were less effective, perhaps due to the degree to which Mg2+ is necessary to support GDP binding (Barren and Artemyev 2007). p21ras forms a tight

2+ complex, while Ga subunits bind Mg2+ in the 2+ 2+ complex. An alterna-

tive approach to make an inhibitor of G protein coupling is to use the C-terminus of Ga which is essential for receptor contact (Gilchrist et al. 2002). Antibodies, peptides, and minigene vectors expressing C-terminal peptides can be used as inhibitors of receptor–G protein interactions resulting in competitive blockade of downstream events. This interaction has been shown to be quite specific with single amino acid changes in the C-terminal peptide resulting in functional differences (Gilchrist et al. 1998).

A similar approach has been used by other investigators to look at a variety of responses related to G proteins including (1) binding of pleckstrin homology (PH) domains to Gbg, (2) inhibiting GPCRs by expressing the C-terminus of b

2- adrenergic receptor kinase, and (3) identifying intracellular domains of GPCRs

critical for G protein coupling. In addition, researchers have made transgenic mice targeting the Ga C-terminus in specific tissues such as the myocardium (DeGeorge et al. 2008) and smooth muscle (McGraw et al. 2007) and the results indicate a marked inhibition of GPCR-mediated responses.


GPCR signaling is also substantially influenced by receptor expression, dimerization, desensitization, and internalization, as well as in response to binding by different ligands and within various cellular contexts. For example, receptor internalization reduces the pool of receptors available for binding thus diminishing signaling when downstream effector pathways are measured.

1.2 Traditional GPCR Pharmacology

1.2.1 Basic Concepts and Language

1.2.1.1 Agonism, Antagonism, Efficacy versus Affinity, Dose–Response Curves, and Drug Receptor Theory

In the most general definition, a drug is any substance that brings about a change in the biologic function of a cell. An agonist binds to and activates the receptor, often mimicking the action of an endogenous ligand (such as a neurotransmitter) that binds to the same receptor. Agonists come in several versions such as full ago-nists which bind and activate a receptor with complete efficacy. An example of a full agonist is isoproterenol which mimics the endogenous ligand epinepherine and acts on b-ARs. Partial agonists are drugs that bind to receptors and activate them but do not evoke as great a response as the full agonists. It is worth mentioning that if both a full agonist (such as the endogenous ligand) and partial agonist are present, the partial agonist will actually serve as an antagonist, competing with the full agonist for receptor occupancy and thus producing a net decrease in the receptor activation relative to that observed with the full agonist alone. Thus, pindolol, which is a partial agonist for b-AR, may act as either a partial agonist (if no full agonist is present) or an antagonist (if a full agonist such as epinephrine is present).

Other drugs act as pharmacological antagonists; i.e., they bind to receptors but do not lead to any signaling by the cell; rather, they interfere with the ability of an agonist to activate the receptor. The effect of a so-called “pure” or “neutral” antago-nist on a cell or in a patient depends entirely on its preventing the binding of agonist molecules and blocking their biologic actions.

An endogenous ligand such as acetylcholine or serotonin may bind and stimu-late receptors that couple to different subsets of G proteins. The apparent promis-cuity allows a ligand to elicit a variety of G protein-dependent responses in different cell types. For instance, the heart may respond to catecholamines (nor-epinephrine and epinepherine) by increasing heart rate by acting on G

s-coupled

b2-ARs while in skin such ligands induce constriction of blood vessels through

Gq-coupled ARs.It is becoming apparent that the relative efficacy of compounds may depend on

the read-out chosen for the assay or the conditions used in the experiment. There


are, in fact, many examples of differences in the pharmacological profile for one receptor when different assay read-outs or conditions are used (Kenakin 2005; Gilchrist 2008).

1.3 And Beyond

1.3.1 Recognition of Inverse Agonists

Because of their underlying nature, inverse agonists (which decrease basal signal-ing) are easier to identify with functional screens versus classical binding assays. In 2004, Terry Kenakin wrote a landmark paper that included a survey of antago-nist–receptor pairings on some 73 GPCR targets. Using results from over a hundred articles he concluded that of the 380 antagonist-receptor pairings, 322 were inverse agonists (85%), while 58 (15%) were neutral antagonists (Kenakin 2004). Somewhat tellingly, all clinically prescribed antagonists were noted to be inverse agonists in the survey. These and other findings have moved inverse agonism to the forefront of receptor theory. However, as was pointed out by Parra and Bond (Parra and Bond 2007), the reclassification does not address the clinical importance of inverse agonism versus neutral antagonism.

Constitutive activity was first shown using recombinant systems, and the validity of this finding was questioned due to the use of a heterologous expression system. The use of recombinant systems has been quite beneficial for drug discovery efforts; however, they have highlighted that GPCRs are not merely on/off switches, but are rather more complex micro-processors. For example, the relative potency profiles for agonists of calcitonin receptor vary depending on whether the receptor is transfected into Chinese hamster ovary (CHO), human embryonic kidney (HEK), or African green monkey kidney (COS) cells. Such variations are probably the result of a long list of phenotypic differences between host cells, including rate of gene transcription, posttranslational transfer, glycosylation, phosphorylation, com-partmentalization, as well as expression levels of intracellular partner components such as G proteins, scaffolding proteins, and accessory proteins (Kenakin 2003).

The a1b

-AR was the first GPCR in which specific point mutations were shown to lead to constitutive receptor activation (Kjelsberg et al. 1992). The discovery of CAMs in the AR family triggered other researchers to look for physiological set-tings in which GPCRs were constitutively active. Many diseases have been shown to be the result of constitutive GPCR activity caused by mutations leading to increased G protein signaling, or upregulated GPCR expression levels (Thompson et al. 2008a).

Serotonin 5-HT receptors are a large family, containing some 14 members. One of these, 5-HT

1A, is a key target for the treatment of mood disorders such as anxiety

and depression. Recently, researchers showed that native 5-HT1A

receptors consti-tutively activate Ga

o in rat brain tissue homogenates (Martel et al. 2007). Moreover,


it has been suggested that alterations in the constitutive activity at the 5-HT2A

and 5-HT

2C receptors are involved in anxiety and depression (Berg et al. 2008).

Histamine receptors are another family that contain constitutively active mem-bers. High-throughput screening (HTS) using a radioligand binding assay with the rat H

3 receptor, and a secondary screening with a guinea pig ileum assay, suggested

species differences in compounds identified as antagonists (Hancock 2006). Repeating the screening with human histamine H

3 receptor led to a number of new

compounds being identified, although the author points out that there are 20 poten-tial variant isoforms with numerous signal transduction pathways, unique distribu-tion patterns, and perhaps differential responses to pharmalogical agents.

When a receptor lacks constitutive activity, an inverse agonist behaves as a neu-tral competitive antagonist. This point was emphasized by a recent report in which what were thought to be neutral antagonists of 5HT

7a were shown to be inverse

agonists (Rauly-Lestienne et al. 2007). Thus, the classification of neutral antagonist should be used cautiously until the constitutive activity of the GPCR has been char-acterized. What seems clear after reviewing numerous papers is a lack of literature in which inverse agonists and neutral antagonists are compared in a clinical setting. Without this direct information, it is difficult to determine if the priority, going forward, should be to screen for neutral antagonists or inverse agonists. This is a critical question given that programs to identify neutral antagonists are often more technically demanding in terms of both screening and medicinal chemistry.

1.3.2 Coming of Age for Allosteric Modulation

GPCRs are naturally allosteric, in that they possess at least two binding sites: one for the endogenous ligand; and one for the G protein. Early observations with GPCRs showed multiple ligand binding states in the absence of GTP, while agonist binding was mostly of a low-affinity form with GTP. The necessity for a distinction between the endogenous ligand binding site on the GPCR and other topographi-cally distinct binding sites led to the terms orthosteric and allosteric, respectively (May and Christopoulos 2003). Drugs that act via the orthosteric site rely on the drug’s affinity, while allosteric modulators are characterized by cooperative bind-ing. Traditionally, modulators of GPCRs (both allosteric and orthosteric) were studied using radioligand binding assays (Tränkle et al. 1999). However, this approach is limited in that it may limit detection of allosteric ligands to those that modulate the affinity of the orthosteric ligand (Kenakin 2006). The advent of func-tional screening using methods such as changes in calcium transients, activation of inositol phosphates and ERK has opened the floodgates for the identification of small molecules that can modulate GPCR function, while having no effect (or even an opposing effect) on agonist binding.

Recently two groups reported identification of small-molecule allosteric ago-nists for the glucagon-like peptide (GLP) receptor (Chen et al. 2007; Knudsen et al. 2007). The work is noteworthy given that no small-molecule agonists had


been previously discovered for any member of the family B GPCR group. In the case of Knudsen et al. none of the 500,000 small molecules served as agonists when assessed by competitive binding assays. The discovery of a substituted qui-noxaline with an IC

50 of 1.4 mM emerged when a cAMP assay was used to screen

250,000 compounds. Additional studies showed the compound increased binding of the radiolabeled peptide ligand, suggesting it might have been identified in competitive binding assays if the screen had initially been set up to look for increased ligand binding. Chen et al. also utilized a cAMP assay to screen GLP receptor, but utilized a cAMP response element (CRE)-driven luciferase reporter plasmid when screening their library of 48,160 compounds. Their assay resulted in the identification of five initial hits of which two compounds were confirmed to increase cAMP levels and displace ligand binding. The use of cAMP and CRE assays is interesting given that some compounds may not necessarily stimulate cAMP responses, but their responses become substantial at the level of CRE-gene transcription (Baker et al. 2004).

The ability to identify allosteric modulators for GPCRs has only emphasized the need to characterize the pharmacology of the ligands in as many assays as pos-sible, given that a lack of effect in one assay does not necessarily translate to the same effect in all systems. Rather, the “efficacy” of a compound be it allosteric or orthosteric is dependent on the assay system in which it is tested (Niedernberg et al. 2003).

1.3.3 Advent of Functional Selectivity

In classical pharmacological terms, intrinsic efficacy is a system-independent parameter that is constant for each ligand at each receptor. Unfortunately for GPCR researchers, this premise is usually incorrect. Recognition that some ligands have diverse functional consequences when they bind led to the hypothesis that each ligand could induce changes in the GPCR resulting in unique conformations. This led to the theory that certain drugs have the ability to preferentially activate one intracellular signaling pathway over another. The phenomenon is known by a num-ber of different terms including “functional selectivity”, “agonist-directed traffick-ing of receptor stimulus”, “biased agonism”, “protean agonism”, “differential engagement”, and “stimulus trafficking”. During the last year researchers have added substantially to the growing body of evidence that functional selectivity is not just a theoretical possibility, but a robust biological phenomenon.

Working with recombinant Gai1 and Ga

o2, researchers revealed significant

differences in the activation patterns of four out of 10 m-opioid receptor agonists (Saidak et al. 2006). While other scientists (Maillet et al. 2007) provided an exam-ple of a compound (LPI805) showing functional selectivity identified in a screen for the tachykinin NK2 receptor. The orthosteric agonist neurokinin A stabilizes the receptor in at least two different active conformations, and LPI805 modulates the relative proportion of the two active conformation states. A first-rate illustration of


functional selectivity was shown in the study of the cortical 5-HT2A

receptor response to hallucinogens (González-Maeso et al. 2007). Another case in point for the extent of functional selectivity used cells that express either b

1-AR or b

2-AR

measuring two distinct signaling pathways (adenylyl cyclase and ERK1/2

) to profile several clinically relevant ligands (Galandrin et al. 2008). The antibody-capture [35S]GTPgS scintillation proximity assay provided evidence of functional selectiv-ity for several GPCRs including the dopamine D

1 receptor (Gazi et al. 2003), and

5-HT1A

receptor (Mannoury la Cour et al. 2006). Taken together, these data are consistent with the idea that different ligands can stabilize distinct receptor confor-mations, and these conformations promote an affinity for one G protein over another, which subsequently govern downstream signaling events (Fig. 1.3).

1.3.4 Where will Dimerization take Us?

GPCRs were initially thought to be monomeric entities, coupling to the G proteins with a 1:1 stoichiometery. Over a decade ago, researchers showed that the GABAB

1

receptor needed to couple to the GABAB2 receptor to traffic to the cell surface

(Kaupmann et al. 1998; White et al. 1998). Since that time, using a number of methods of detection including co-immunoprecipitation, FRET and biolumines-cence resonance energy transfer (BRET), an increasing number of GPCRs have

ARR2 GIP

R R R

αβγ

G proteindependentsignaling

Endocytosis G proteinindependentsignaling

EndocytosisG proteinindependentsignaling

G proteindependentsignaling

ARR2ARR1α

βγ

Fig. 1.3 Functional selectivity can occur with GPCRs. The conformation of GPCRs appears to be ligand-dependent, and different agonists can produce selective G protein dependent or indepen-dent signaling. Certain ligands may have functional selectivity towards responses not mediated through G proteins, but rather signal via arrestins or other GPCR-interacting proteins (GIPs). Which messenger proteins first become activated as a result of GPCR/ligand interaction influence the downstream effectors employed. Thus, signaling via G proteins, arrestins, or GIPs are critical in determining cellular responses


been shown to be expressed at the plasma membranes as homodimers, heterodim-ers, or higher order oligomers (Gurevich and Gurevich 2008). These “horizontal molecular networks” (Agnati et al. 2005) may also contain other interacting pro-teins. While the functional significance of receptor dimerization is still not com-pletely understood, and in some cases even the applied methodological approaches are debated, it is now generally accepted that oligomerization of GPCRs not only exists, but that it is relevant to receptor expression and function, including agonist binding, potency, and efficacy (Szidonya et al. 2008). An open question still remains on whether there is a requirement for the receptor to be dimeric for G protein activation (White et al. 2007; Damian et al. 2008).

As a heterodimer, the receptors may display a different conformation, and thus may have a pharmalogical profile (ligand binding and/or G protein coupling) that is distinct from the receptor monomer or homodimer. For example the D

2 receptor

normally couples to Gi/o

proteins; however, in the D1–D

2 heterodimer it switches

to Gq/11

when D1 is co-activated (Lee et al. 2004; Rashid et al. 2007). New equa-

tions to fit the binding data for receptor homodimers have been introduced (Giraldo 2008). In addition to providing equilibrium dissociation constants for binding of the first and second molecule (low- and high-affinity, respectively) to the dimer, they provide a measure of the dimer cooperativity index. No doubt similar equations for fitting binding data for heterodimer formations will soon be determined as well.

It is important to point out that in vivo, receptors form heteromers that differ depending on the cell type, and pharmacological differences in antagonist activity/affinity depending on the partner protein have been noted. This adds a new level of complexity to drug development, but reveals a new arena in which drugs can be designed to bind preferentially to one dimer formation over another. Another novel therapeutic approach for dimers is the design of dimeric compounds that act on both receptors in the heterodimer complex (Waldhoer et al. 2005; Rashid et al. 2007).

1.3.5 Quantifying Drug Activity

One of the most well known models for GPCRs is the two-state model of receptor activation, which postulates that there are at least two conformational forms that are in equilibrium: R and R* (Leff 1995). In this model R* is capable of signaling, and it is considered the “active” molecule, while R is considered to be the inactive state of the receptor. More complex models which include allosteric sites such as the “ternary complex model” proposed by De Lean et al. (De Lean et al. 1980) include an allosteric or regulatory site where G proteins can bind. Samama and colleagues (Samama et al. 1993) expanded this model and developed the “extended ternary complex model,” which includes different affinity states for the receptor uncoupled and coupled to the G protein, R and R*, respectively. From this the “allosteric two-state model” was developed in which allosteric modulation was not restricted to


G proteins (Hall 2000). More recently, the “quaternary complex model” of allos-teric interactions have been proposed (Christopoulos and Kenakin 2002).

The use of radiolabeled ligands and binding assays to determine binding con-stants (association, K

A, or dissociation, K

D = 1/K

A) has proven to be an invaluable

tool for screening GPCRs for potential drugs. Curve fitting of experimental data points by mathematical models is a common way to extract relevant information from biological systems being regulated by GPCRs. Mathematical models are classified as mechanistic or empirical. Mechanistic models are the ideal formu-lations for the analysis of experimental curve data points, and can lead to new knowledge of the underlying biological mechanisms. Unfortunately, they often have a high number of parameters precluding their use by classical curve-fitting procedures such as gradient nonlinear regression. Empirical models employ the minimum number of parameters for the determination of the shape of the curve and, accordingly, do not present difficulties for standard curve fitting. In general, empirical models lack physical basis and are limited to obtaining the common geometric descriptors (midpoint location and slope, asymptotes, etc.) of the curves.

Over the years the pharmaceutical industry has shifted away from ligand binding assays to functional assays for their large primary screens of GPCRs. The reasons for the change include a desire to decrease the use of radioactivity, reduce the cost of screening their large (hundreds of thousands) compound libraries, and identify allosteric modulators targeting sites independent from the orthosteric ligand binding site. The initial functional assays employed were focused on well-validated second messenger signaling pathways such as calcium, and cAMP. New assays that look to alternatives in the downstream signaling cascade have been introduced, including IP

3 (Trinquet et al. 2006) and ERK (Osmond et al. 2005; Leroy et al. 2007). ERK

1/2

phosphorylation assay offers an advantage in that it is a convergent signaling point, and its activation can be both G protein dependent and independent. More recent methods of screening for small-molecule modulators of GPCRs include cellular dielectric spectroscopy, flow cytometry, capillary electrophoresis, electronic bio-sensors, and the use of genetic selection in yeast (Gilchrist 2008). There has also been a trend towards adapting high content screening (HCS) approaches for GPCRs (Henriksen et al. 2008; Ross et al. 2008).

Another interesting GPCR screening trend was the advent of b-arrestin screen-ing approaches (Ghosh et al. 2005; Hammer et al. 2007). Arrestins are a family of four GPCR-binding proteins that play a key role in homologous desensitization and endocytosis (Defea 2008). They were believed to play a role only in limiting GPCR signaling by binding the GPCR and physically interfering with the binding of the G protein. However, scientists have now demonstrated that b-arrestin signaling extends beyond the regulation of G protein pathways and receptor trafficking and suggest that G proteins and arrestins link GPCRs to distinct sets of downstream effectors capable of eliciting both redundant and unique cellular responses (DeWire et al. 2007). In fact, recent evidence indicates that certain ligands can selectively activate b-arrestin signaling independent of G protein signaling (Violin and Lefkowitz 2007). For example, the parathyroid hormone (PTH) analog (D-Trp12,


Tyr34) PTH(7–34) acts as an inverse agonist for PTH1 receptor–Gs coupling while

promoting arrestin-dependent sequestration (Gesty-Palmer et al. 2006). Similarly, ICI118551, propranolol, and carvedilol, b

2-AR ligands that act as inverse agonists

with respect to Gs activation, function as partial agonists for the ERK

1/2 pathway by

engaging the b-arrestin (Azzi et al. 2003; Wisler et al. 2007). The opposite is also possible, such that Trp1-PTHrP(1–36), another PTH analog, promotes G

s coupling

without inducing b-arrestin recruitment or desensitization (Gesty-Palmer et al. 2006). Such findings support the idea that GPCRs are capable of assuming multiple conformations, but the clinical utility of functionally selective compounds will ultimately depend on whether inducing only a subset of the full response profile confers therapeutic benefits.

Work in the field of functional selectivity has made it clear that a compound’s efficacy is ultimately defined in terms of the assay used to measure it, as com-pounds that were disregarded on the basis of apparently poor efficacy for one response (i.e., calcium) may be found to be effective if other assays are employed (i.e., ERK

1/2). Researchers also now recognize the importance of the “environment”

for the GPCRs during the drug discovery process as changes in the GPCR expres-sion level, G proteins present, G protein:GPCR ratio, as well as the availability of accessory proteins all influence the efficacy of the drugs being tested (Eglen et al. 2008; Gilchrist 2008).

The transduction of many diverse signals from the extracellular to intracellular environments requires the actions of GPCRs, and at the same time, mutations in GPCRs underlie a variety of human diseases (Thompson et al. 2008b). However, in spite of the advances made by researchers in past years, the answers to many fun-damental questions remain unanswered, including how GPCR activation through ligand binding promotes GDP–GTP exchange on Ga, and defining the critical role that Gbg plays in this process. It will also be important to discern how binding events at the orthosteric and allosteric binding sites mechanistically lead to their downstream events. Many of the answers will require a better understanding of the how, when, and why specific heterotrimeric G proteins or alternative GPCR signal-ing partners get activated.

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