ENDOCRINOLOGY · 2013-07-18 · endocrinology. The challenge in its presentation was to produce a...

ENDOCRINOLOGY

ENDOCRINOLOGYBasic and Clinical PrinciplesSECOND EDITION

Edited by

SHLOMO MELMED, MDCedars Sinai Medical Centerand UCLA School of Medicine

P. MICHAEL CONN, PhDOregon Health & Science UniversityBeaverton, OR

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Library of Congress Cataloging-in-Publication DataEndocrinology : basic and clinical principles / edited by Shlomo Melmed, P. Michael Conn.— 2nd ed. p. cm. Includes bibliographical references and index. ISBN 1-58829-427-7 (alk. paper) eISBN 1-59259-829-3 1. Endocrinology. 2. Hormones. 3. Endocrine glands—Diseases. I. Melmed, Shlomo. II. Conn, P. Michael. QP187.E555 2005 612.4—dc22 2004018638

v

Endocrinology: Basic and Clinical Principles, Sec-ond Edition aims to provide a comprehensive knowl-edge base for the applied and clinical science ofendocrinology. The challenge in its presentation wasto produce a volume that was timely, provided integra-tion of basic science with physiologic and clinical prin-ciples, and yet was limited to 500 pages. This lengthmakes the volume suitable as a text; and the timelinesswe have striven for allows the book to serve as an off-the-shelf reference. Our goal was achieved largelythrough the selection of authors who are both expertwriters and teachers. Tables and illustrative matterwere used optimally to present information in a con-cise and comparative format.

Endocrinology: Basic and Clinical Principles, Sec-ond Edition will be useful to physicians and scientists aswell as to students who wish to have a high-quality,

PREFACE

current reference to the general field of endocrinology.The use of an outline system and a comprehensive indexwill allow readers to locate promptly topics of particu-lar interest. Key references are provided throughout forindividuals requiring more in-depth information. Thevolume covers the comprehensive spectrum of currentknowledge of hormone production and action, evenincluding nonmammalian systems and plants, coveragerarely included in similar volumes.

The editors wish to express appreciation to our dis-tinguished chapter authors for their efforts, as well asdiligently meeting publication deadlines and to the staffat Humana Press for their cooperation and useful sug-gestions.

Shlomo MelmedP. Michael Conn

Preface .................................................................................................................................. vii

Contributors ........................................................................................................................... ix

Value Added eBook ...............................................................................................................xi

PART I. INTRODUCTION

1 Introduction to Endocrinology .................................................................................... 3

P. Michael Conn

PART II. HORMONE SECRETION AND ACTION2 Receptors: Molecular Mediators of Hormone Action ................................................... 9

Kelly E. Mayo

3 Second-Messenger Systemsand Signal Transduction Mechanisms .................................................................. 35

Eliot R. Spindel

4 Steroid Hormones......................................................................................................... 49

Derek Henley, Jonathan Lindzey, and Kenneth S. Korach

5 Plasma Membrane Receptors for Steroid Hormonesin Cell Signaling and Nuclear Function ................................................................ 67

Richard J. Pietras and Clara M. Szego

6 Growth Factors ............................................................................................................. 85

Derek LeRoith and William L. Lowe Jr.

7 Prostaglandins and Leukotrienes: Locally Acting Agents ........................................... 93

John A. McCracken

8 The Neuroendocrine–Immune Interface .................................................................... 113

Michael S. Harbuz and Stafford L. Lightman

Part III. Insects/Plants/Comparative9 Insect Hormones ......................................................................................................... 127

Lawrence I. Gilbert

10 Phytohormones and Signal Transduction Pathways in Plants .................................. 137

William Teale, Ivan Paponov, Olaf Tietz, and Klaus Palme

11 Comparative Endocrinology ...................................................................................... 149

Fredrick Stormshak

vii

CONTENTS

Part IV. Hypothalamic–Pituitary12 Hypothalamic Hormones:

GnRH, TRH, GHRH, SRIF, CRH, and Dopamine ..............................................173Constantine A. Stratakis and George P. Chrousos

13 Anterior Pituitary Hormones ..................................................................................... 197Ilan Shimon and Shlomo Melmed

14 Posterior Pituitary Hormones .................................................................................... 211Daniel G. Bichet

15 Endocrine Disease: Value for Understanding Hormonal Actions ............................233

Anthony P. Heaney and Glenn D. Braunstein

16 The Pineal Hormone (Melatonin) .............................................................................. 255

Irina V. Zhdanova and Richard J. Wurtman

17 Thyroid Hormones (T4, T

3) ....................................................................................... 267

Takahiko Kogai and Gregory A. Brent

18 Calcium-Regulating Hormones:Vitamin D and Parathyroid Hormone ................................................................283

Geoffrey N. Hendy

19 Oncogenes and Tumor Suppressor Genes in Tumorigenesisof the Endocrine System...................................................................................... 301

Anthony P. Heaney and Shlomo Melmed

20 Insulin Secretion and Action ..................................................................................... 311

Run Yu, Hongxiang Hui, and Shlomo Melmed

21 Cardiovascular Hormones .......................................................................................... 321

Willis K. Samson and Meghan M. Taylor

22 Adrenal Medulla (Catecholamines and Peptides) ..................................................... 337

William J. Raum

23 Hormones of the Kidney ............................................................................................ 353

Masashi Mukoyama and Kazuwa Nakao

24 Reproduction and Fertility ......................................................................................... 367

Neena B. Schwartz

25 Endocrinology of Fat, Metabolism, and Appetite ..................................................... 375

Rachel L. Batterham and Michael A. Cowley

26 Endocrinology of the Ovary ...................................................................................... 391

Denis Magoffin, Ashim Kumar, Bulent Yildiz, and Ricardo Azziz

27 The Testis ................................................................................................................... 405

Amiya Sinha Hikim, Ronald S. Swerdloff, and Christina Wang

28 Endocrinology of Aging ............................................................................................ 419Steven W. J. Lamberts

Index .................................................................................................................................... 429

Contents viii

ix

RICARDO AZZIZ, MD, MPH, MBA • Department ofObstetrics and Gynecology, Cedars-Sinai MedicalCenter, UCLA School of Medicine, Los Angeles, CA

RACHEL L. BATTERHAM, MBBS, PhD • University CollegeLondon, London, UK

DANIEL G. BICHET, MD • Clinical Research Unit andNephrology Service, Hospital du Sacre-Coeur deMontreal, Montreal, Canada

GLENN D. BRAUNSTEIN, MD • Department of Medicine,Cedars-Sinai Medical Center, UCLA Schoolof Medicine, Los Angeles, CA

GREGORY A. BRENT, MD • Departments of Medicineand Physiology, UCLA School of Medicine,Los Angeles, CA

GEORGE P. CHROUSOS, PhD • National Institute of ChildHealth & Human Development, National Institutesof Health, Bethesda, MD

P. MICHAEL CONN, PhD • Oregon National PrimateResearch Center, Oregon Health and ScienceUniversity, Beaverton, OR

MICHAEL A. COWLEY, PhD • Oregon National PrimateResearch Center, Oregon Health and ScienceUniversity, Beaverton, OR

LAWRENCE I. GILBERT, PhD • Department of Biology,University of North Carolina, Chapel Hill, NC

MICHAEL S. HARBUZ, PhD • Department of Medicine,Henry Wellcome Laboratories for IntegratedNeuroscience and Endocrinology, Universityof Bristol, Bristol, UK

ANTHONY P. HEANEY, MD, PhD • Division ofEndocrinology and Metabolism, Cedars-SinaiMedical Center, UCLA School of Medicine, LosAngeles, CA

GEOFFREY N. HENDY, PhD • Department of Medicine,Royal Victoria Hospital, McGill University,Montreal, Canada

DEREK V. HENLEY, PhD • Laboratory of Reproductiveand Developmental Toxicology, National Instituteof Environmental Health Sciences, NationalInstitutes of Health, Research Triangle Park, NC

AMIYA SINHA HIKIM, PhD • Division of Endocrinology,Department of Medicine, Harbor-UCLA MedicalCenter and Education and Research Institute,UCLA School of Medicine, Torrance, CA

CONTRIBUTORS

HONGXIANG HUI, MD, PhD • Division of Endocrinology,Cedars-Sinai Medical Center, UCLA Schoolof Medicine, Los Angeles, CA

TAKAHIKO KOGAI, MD, PhD • Division of Endocrinologyand Diabetes, VA Greater Los Angeles HealthcareSystem, UCLA School of Medicine,Los Angeles, CA

KENNETH S. KORACH, PhD • Laboratory of Reproductiveand Developmental Toxicology, National Instituteof Environmental Health Sciences, NationalInstitutes of Health, Research Triangle Park, NC

ASHIM KUMAR, MD • Department of Obstetrics andGynecology, Cedars-Sinai Medical Center, UCLASchool of Medicine, Los Angeles, CA

STEVEN W. J. LAMBERTS, MD, PhD • Departmentof Medicine, Erasmus Medical Center, Rotterdam,The Netherlands

DEREK LEROITH, MD, PhD • Diabetes Branch, NationalInstitutes of Health, Bethesda, MA

STAFFORD L. LIGHTMAN, PhD • Department of Medicine,Henry Wellcome Laboratories for IntegratedNeuroscience and Endocrinology, Universityof Bristol, Bristol, UK

JONATHAN LINDZEY, PhD • Department of NaturalSciences, Clayton College and State University,Morrow, GA

WILLIAM L. LOWE JR., MD • Department of Medicine,Northwestern University Medical School,Chicago, IL

DENIS MAGOFFIN, PhD • Department of Obstetrics andGynecology, Cedars-Sinai Medical Center, UCLASchool of Medicine, Los Angeles, CA

KELLY E. MAYO, PhD • Department of Biochemistry,Molecular Biology, and Cell Biology, Centerfor Reproductive Science, Northwestern University,Evanston, IL

JOHN A. MCCRACKEN, PhD • University of Connecticut,Storrs, CT

SHLOMO MELMED, MD • Division of Endocrinology andMetabolism, Cedars-Sinai Medical Center, UCLASchool of Medicine, Los Angeles, CA

MASASHI MUKOYAMA, MD, PhD • Department of Medicineand Clinical Science, Kyoto University GraduateSchool of Medicine, Kyoto, Japan

KAZUWA NAKAO, MD, PhD • Department of Medicineand Clinical Science, Kyoto University Graduate Schoolof Medicine, Kyoto, Japan

KLAUS PALME, PhD • Institute for Biology,Albert-Ludwigs-Universität Freiburg, Freiburg,Germany

IVAN PAPONOV, PhD • Institute for Biology,Albert-Ludwigs-Universität Freiburg, Freiburg,Germany

RICHARD J. PIETRAS, MD, PhD • Departmentof Medicine, Division of Hematology-Oncology,Jonsson Comprehensive Cancer Center, UCLASchool of Medicine, Los Angeles, CA

WILLIAM J. RAUM, MD, PhD • Departments of Medicineand Surgery, Louisiana State University MedicalCenter, New Orleans, LA

WILLIS K. SAMSON, PhD • Department ofPharmacologic and Physiologic Science, St.Louis University School of Medicine, St. Louis,MO

NEENA B. SCHWARTZ, PhD • Department of Neuro-biology and Physiology, Northwestern University,Evanston, IL

ILAN SHIMON, MD • Institute of Endocrinology, ShebaMedical Center, Tel-Hashomer, Israel

ELIOT R. SPINDEL, MD, PhD • Oregon National PrimateResearch Center, Oregon Health and ScienceUniversity, Beaverton, OR

FREDRICK STORMSHAK, PhD • Departments ofBiochemistry/Biophysics and Animal Sciences,Oregon State University, Corvalis, OR

CONSTANTINE A. STRATAKIS, PhD, DSc • NationalInstitutes of Health, Bethesda, MD

RONALD S. SWERDLOFF, MD • Division of Endocrinology,Department of Medicine, Harbor-UCLA MedicalCenter and Education and Research Institute,UCLA School of Medicine, Torrance, CA

CLARA M. SZEGO, PhD • Department of Molecular,Cellular, and Developmental Biology, MolecularBiology Institute, University of California, LosAngeles, CA

MEGHAN M. TAYLOR, PhD • Department of Pharma-cologic and Physiologic Science, St. LouisUniversity School of Medicine, St. Louis, MO

WILLIAM TEALE, PhD • Institute for Biology, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany

OLAF TIETZ, PhD • Institute for Biology, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany

CHRISTINA WANG, MD • Division of Endocrinology,Department of Medicine, General ClinicalResearch Center, Harbor-UCLA Medical Centerand Education and Research Institute, UCLASchool of Medicine, Torrance, CA

RICHARD J. WURTMAN, MD • Department of Brainand Cognitive Sciences, Clinical Research Center,Massachusetts Institute of Technology,Cambridge, MA

BULENT YILDIZ, MD • Interdepartmental ClinicalPharma-cology Center, UCLA School of Medicine,Los Angeles, CA

RUN YU, MD, PhD • Division of Endocrinology, Cedars-Sinai Medical Center, UCLA School of Medicine, LosAngeles, CA

IRINA V. ZHDANOVA, MD, PhD • Department of Anatomyand Neurobiology, Boston University Schoolof Medicine, Boston, MA

x Contributors

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xi

Chapter 1 / Short Chapter Title 1

INTRODUCTIONPART

I


3

From: Endocrinology: Basic and Clinical Principles, Second Edition(S. Melmed and P. M. Conn, eds.) © Humana Press Inc., Totowa, NJ

1 Introduction to Endocrinology

P. Michael Conn, PhD

CONTENTS

INTRODUCTION

DEFINITIONS

HORMONES CONVEY INFORMATION THAT REGULATES CELL PROCESSES

IDENTIFYING HORMONES

HORMONE-DERIVED DRUGS

ENDOCRINOLOGY AS A LEAD SCIENCE

responses in another animal are referred to as phero-mones.

Sometimes the word hormone is used as a referenceto substances in plants (phytohormones) or in inverte-brates that have open “circulatory” systems very dif-ferent from those found in vertebrates. On otheroccasions, growth factors are (appropriately) calledhormones, because they mediate signaling betweencells. In recent years, the word has become a catchall todescribe substances released by one cell that provoke aresponse in another cell even when the messenger sub-stance does not enter the general circulation. The sci-ence of endocrinology has broad coverage indeed.

3. HORMONES CONVEY INFORMATIONTHAT REGULATES CELL PROCESSES

Characteristically, hormones transmit informationabout the status of one organ to another, regulating cor-rective actions to maintain homeostasis. For example,elevated glucose in the blood signals the pancreas torelease insulin. Insulin travels through the circulation,signaling target cells in liver and fat cells to increasetheir permeability to glucose; conversely, processedsugar is stored in cells as blood levels drop.

1. INTRODUCTIONThe earliest bacterial fossils date back about 3 bil-

lion years. That was a simpler time! Communicationsbetween cells were more modest than those required tomaintain a multicellular organism and were probablyfocused on the ability to signal the presence of benefi-cial substances (food) or deleterious substances (tox-ins) in the local environment.

2. DEFINITIONSSubstances that provide the chemical basis for com-

munication between cells are called hormones. Thisword, coined by Bayliss and Starling, was originallyused to describe the products of ductless glandsreleased into the general circulation in order to respondto changes in homeostasis. Hormone has taken on abroader usage in recent years. Sometimes hormonesare released into portal (closed) circulatory systemsand have local actions. The word paracrine is used todescribe the release of locally acting substances. Thisword also describes local hormone action as the diffu-sion of gastric juice acts on neighboring cells. Hor-monal substances released by an animal that influence

4 Part I / Introduction

To be effective, hormones should not be degradedtoo quickly (i.e., before arrival at the target site). Ifdegradation is too slow, on the other hand, the infor-mation conveyed will be obsolete and may evoke aninappropriate response. Accordingly, it is not surpris-ing that different hormones have varying half-lives inthe circulation, depending, in part, on the distance thatthe signal must travel and the nature of the informationto be conveyed.

Concentrations of hormones are sensed by recep-tors, usually proteins, located on the surface (i.e.,plasma membrane) or inside target cells. Receptorsbind their respective hormone ligands with high affin-ity and specificity. For example, although estrogen andtestosterone are chemically similar, receptors must dis-tinguish between them because they mediate very dif-ferent cellular responses indeed. When hormonereceptors are situated on the surface of target cells andthe response involves intracellular changes (e.g., evok-ing secretione), transduction of the hormonal messagemust occur. Such transduction molecules are termedsecond messengers of hormone action.

It is a general truth that the chemical structures ofhormones do not change markedly during evolution;instead, nature identifies and conserves molecules thatalready have information value and develops systemsthat preserve and utilize that information. Steroids, thy-roid hormones, and peptides are present in some speciesthat do not utilize them for the same endocrine purposeas do mammals.

4. IDENTIFYING HORMONESThe effects of ablation of endocrine organs have

been documented back to the time of Aristotle (384–322 BC), who described changes in secondary sex char-acteristics and loss of reproductive capacity associatedwith castration in men. Much insight into the role ofendocrine substances has come from disease states,surgical errors, and animal experimentation in whichdamage to endocrine organs is correlated with particu-lar phenotypic changes in the organism.

Ancient medical procedures prevalent in many cul-tures were based on the premise that administration ofextracts from healthy organs aids in the recovery of dis-eased organs. This practice may be viewed as a prede-cessor to hormone replacement therapy. Restoration offunction by supplements derived from healthy endo-crine organs administered to animals with endocrineablations has formed the basis of discovering activeprinciples of the endocrine system.

In the mid-1800s, Berthold showed that the effects ofcastration in avians could be reversed by placing a testisin the body cavity. Since the transplant was ectopic and

not innervated, he concluded that the testes released asubstance that controlled secondary sex characteristics.

A few years later, Claude Bernard, providing evi-dence to support a model of homeostasis, showed thatthe liver could release sugar to the blood. From the mid-1850s to the twentieth century, endocrinology grew at adramatic pace. Assays became more sensitive and spe-cific; biosynthetic and genetic engineering techniquesnow allow synthesis of biologically active and highlypurified hormones.

5. HORMONE-DERIVED DRUGSThe identification of new hormonal activities often

follows a similar pattern. The observation is made thatdamage to a particular gland is associated with loss of acertain function. Efforts are then focused on isolatingthe active principle from the gland. The active principleis then administered to restore the function to the animalpatient who has ablated glandular function. The devel-opment of drugs is usually directed toward preparingpurified fractions that can be used in replacementtherapy. The hormone itself and, ultimately, chemicalanalogs can now readily be synthesized. Analogs can bedesigned to possess desirable properties, such as pro-longed circulation half-lives, chemical stability, or spe-cific receptor or tissue targeting. The availability ofpurified fractions or synthetic hormone preparationsoften spawns studies designed to understand the cellularand molecular basis of hormone action. This informa-tion is then used to design even more useful drugs thatrecognize the target cell receptor with higher specificityand affinity; antagonists can also be prepared that blockthe receptor or its signaling. The science of endocrinol-ogy is poised to take advantage of our understanding ofintricate second-messenger systems, sensitive and pre-cise assay systems (radioimmunoassays, bioassays,radioligand assays), and advances in structural and func-tional molecular biology. As the tools of endocrinologyhave become more precise, we have discovered that eventhe brain, heart, and lung possess substantial endocrinefunctions.

6. ENDOCRINOLOGYAS A LEAD SCIENCE

Endocrinology continues to be a lead science. ManyNobel Prizes have recognized the contributions of endo-crinologists. The first cloned gene products to reach theclinical pharmaceutical market were endocrine sub-stances. Many advances in our understanding of cellulartransduction systems, receptor binding, and physiologicregulation are derivatives of the studies conducted inendocrine laboratories. Why is this so? A likely answer


is found by understanding that endocrinologists studythe actions of specific chemicals that cause cells toundergo specific and (usually) easily quantifiable andregulated responses. These are very simple, basic, andwell-defined processes. Accordingly, clear and inter-pretable experiments can be designed at a complexity

ranging from molecular to physiologic. This is part ofthe general appeal and high level of achievement of thisscience—and much of the reason that those who callthemselves endocrinologists have made a major contri-bution to our understanding of regulatory biologic pro-cesses.

Chapter 2 / Receptors 7

HORMONE SECRETION AND ACTIONPART

II


9

From: Endocrinology: Basic and Clinical Principles, Second Edition(S. Melmed and P. M. Conn, eds.) © Humana Press Inc., Totowa, NJ

2 ReceptorsMolecular Mediators of Hormone Action

Kelly E. Mayo, PhD

CONTENTS

INTRODUCTION

GENERAL ASPECTS OF RECEPTOR ACTION

CELL-SURFACE RECEPTORS

INTRACELLULAR RECEPTORS

RECEPTORS, HEALTH, AND DISEASE

CONCLUSION

1. INTRODUCTIONThe appropriate proliferation and differentiation of

cells during development and the maintenance of cellu-lar homeostasis in the adult require a continuous flow ofinformation to the cell. This is provided either by diffus-ible signaling molecules or by direct cell–cell and cell–matrix interactions. All cells utilize a wide variety ofsignaling molecules and signal transduction systems tocommunicate with one another, but within the verte-brate endocrine system, it is the secreted hormones thatare classically associated with cellular signaling. Hor-mones are chemical messengers produced from the en-docrine glands that act either locally or at a distance toregulate the activity of a target cell. As discussed indetail elsewhere within this volume, prominent groupsof hormonal agents include peptide hormones; steroid,retinoid, and thyroid hormones; growth factors; cyto-kines; pheromones; and neurotransmitters or neuro-modulators.

Endocrine signaling molecules exert their effects byinteracting with specific receptor proteins that are gen-erally coupled to one or more intracellular effector sys-

tems. The presence of an appropriate receptor thereforedefines the population of target cells for a given hor-mone and provides a molecular mechanism by whichthe hormone elicits its biologic actions. These hormonereceptor proteins are the focus of this chapter. Section 2considers general concepts of receptor action, includingreceptor structure, interaction with the hormone ligand,activation of cellular effector systems, and receptorregulation. Sections 3 and 4 then examine the majorfamilies of hormone receptors, grouped with respectto their structures and signaling properties, in greaterdetail, using specific examples that illustrate the generalfeatures of each family. Finally, Section 5 discussessome of the endocrinopathies that result from knownalterations in hormone receptor structure or function.

2. GENERAL ASPECTSOF RECEPTOR ACTION

2.1. Receptors as Mediatorsof Endocrine Signals

The concept that hormone action is mediated byreceptors is most often attributed to the work of Langleyon the actions of nicotine and curare on the neuromus-

10 Part II / Hormone Secretion and Action

cular junction (Langley, 1906). Langley referred to thetarget for these compounds as the “receptive substance,”and postulated that “it receives the stimulus, and bytransmitting it, causes contraction,” a clear descriptionof the role of receptors in mediating the actions of sig-naling molecules. Of course, Langley could not knowthe nature of these receptive substances, suggesting onlythat they were “radicals of the protoplasmic molecule.”Indeed, despite a wealth of physiologic evidence insupport of the receptor concept, firm biochemical evi-dence for the existence of specific receptors was notforthcoming until radiolabeled hormones became avail-able. For hormones unable to traverse the cellular mem-brane, such as the polypeptide hormones, specificbinding sites could be demonstrated, but their mem-brane association and low abundance precluded theircharacterization for many years. Only with the adventof molecular cloning techniques was it possible toestablish firmly the structures of the hormone receptors,and to show that these proteins could, in effect, converta nontarget cell into a target cell by conferring to the cellthe ability to bind and, in some cases, appropriatelyrespond to the corresponding hormone.

A second key event in the development of the recep-tor concept was the demonstration by Sutherland (1972)that cyclic adenosine monophosphate (cAMP) couldmediate the intracellular effects of many different hor-mones, establishing the notion of “second messengers”and providing a cogent molecular explanation of howhormones and hormone receptors might elicit theirwidespread effects on cellular activity. The notion thatmany different signaling molecules, working throughdistinct receptors, could stimulate a common intracellu-lar signaling pathway, together with the subsequent dis-covery of additional effector enzymes and intracellularsecond messengers, provided a rational explanation forthe tremendous diversity in cellular responses to hor-mones.

At about the time that second-messenger systemswere being discovered, different concepts were evolv-ing on the mechanism of action of the small lipophilicsteroid hormones. Radiolabeled steroids, such as estro-gen, were synthesized by Jensen and others and werefound to accumulate preferentially in known targetorgans for the hormone (Jensen and Jacobson, 1962).Specific steroid-binding proteins were subsequentlyidentified, and the important finding that binding ofagonists and antagonists to these receptors correlatedwith their biologic activity provided evidence that theywere direct mediators of steroid action. These steroidreceptors were shown to be soluble intracellular pro-teins, differentiating them from the membrane-associ-ated receptors for the polypeptide hormones, and

facilitating their early purification, beginning with theidentification of the estrogen receptor by Toft andGorski (1966). The findings that the steroid receptorsassociated with chromatin in the cell nucleus and thatsteroid hormones affected RNA and protein synthesisprovided the basis for models of direct genomic actionsof the hormone-receptor complex that we now know tobe correct.

2.2. Membrane-Associatedvs Intracellular Receptors

Substances that act as signaling molecules haveextremely diverse structures and chemical properties.They include complex multisubunit proteins such asfollicle-stimulating hormone (FSH), small peptidessuch as somatostatin, steroids such as estrogen, aminoacid derivatives such as thyroid hormone or the cat-echolamines, vitamin derivatives such as the retinoidsor calcitrol, fatty acid derivatives such as prostaglan-dins, and simple compounds such as nitric oxide (NO),to provide but a few examples. Despite this structuralcomplexity, most hormones act on target cells throughone of two basic mechanisms. Small lipophilic mol-ecules such as the steroids, retinoids, calcitrol, and thy-roid hormone are able to enter cells directly bydiffusion across the lipid bilayer, where they bind tointracellular receptors that mediate their subsequentaction by directly regulating gene expression. By con-trast, hydrophilic molecules such as the protein andpeptide hormones must bind to their receptor at the cellsurface, and the receptors are therefore typically inte-gral membrane proteins that activate intracellular sig-nal transduction cascades. Thus, it is common todifferentiate between membrane-bound and intracel-lular receptors when considering mechanisms of hor-mone action.

The consequence of hormone-receptor interactionis the activation of pathways that lead to an appropriatebiologic response in the target cell, an area referred toas a signal transduction, which is discussed in depth inChapter 3. Briefly, most intracellular receptors, suchas the steroid hormone receptors, are ligand-activatedtranscription factors that directly regulate the tran-scriptional activity of target genes. By contrast, cell-surface receptors transduce a signal to the cell interior,directly or indirectly altering the activity of proteinswithin the cell. This action often involves the enzy-matic generation of a second messenger, and theactivation of protein phosphorylation or dephosphory-lation cascades. More important, the eventual conse-quence of the activation of enzymatic signalingpathways by cell-surface receptors is often a change ingene transcription; conversely, changes in enzymatic


activities often follow as a consequence of altered geneactivity when intracellular receptors are activated. Fur-thermore, there are many emerging examples of “crosstalk” between cell-signaling pathways (Dumont et al.,2001). Finally, as our understanding of the complexityof receptor signaling increases, exceptions to thesegeneral modes of action of membrane-bound vs intra-cellular receptors are being identified. Thus, many ofthe historical mechanistic boundaries between cell-surface and intracellular receptors are breaking down.Nonetheless, these categories provide a useful way oforganizing thinking about receptor action and aretherefore used to classify the receptors in this chapter.Figure 1 illustrates some of the similarities and differ-ences in hormone signaling through intracellular vscell-surface receptors.

The membrane-associated receptors are an extremelydiverse group of signaling molecules. Their commonfeature is the presence of one or more hydrophobic

domains that span the cell membrane and anchor thereceptor at the cell surface. Extracellular regions of theprotein often participate in hormone binding, and intra-cellular sequences generally either have direct enzy-matic function or associate with intermediary proteinsor effector enzymes. The functional cell-surface hor-mone receptor is often composed of multiple proteinsubunits that may play a role in hormone binding, sig-naling, or both activities. It is clear that the receptors forstructurally and functionally diverse hormones can beclosely related to one another, most commonly withintheir intracellular domains. Thus, a limited number ofbasic signaling strategies are used repetitively by avery large number of receptor proteins, defining distinctreceptor families, which are considered in Section 3.

The intracellular receptors for the steroid hormoneswere the first to be biochemically purified. Followingthe molecular cloning of the receptors for all of the clas-sic steroid hormones (Evans, 1988) it became clear that

Fig 1. Hormonal signaling by cell-surface and intracellular receptors. Protein hormones (P) bind to cell-surface receptors that activateintracellular effector enzymes, often leading to the production of second messengers or the initiation of protein phosphorylationcascades. A longer-term effect is gene transcription and new protein synthesis. By contrast, steroid hormones (S), or other lipophilicligands, bind to intracellular receptors that are ligand-activated transcription factors (TF) that directly mediate gene transcription,leading to new protein synthesis.


these receptors comprise a family of highly related pro-teins consisting of distinct structural domains. Thehighly conserved central DNA-binding domain (DBD)is generally flanked by a carboxyl-terminal region thatis necessary for hormone binding by one or more addi-tional domains that are important for activation of targetgene transcription. The subsequent identification of thethyroid hormone, retinoic acid, and vitamin D receptorsplaced them in this nuclear receptor superfamily, alongwith an expanding number of “orphan receptors” thatact in a ligand-independent fashion or for which appro-priate ligands have not yet been identified (Willson andMoore, 2002). Not all intracellular receptors are struc-turally related to the steroid hormone receptors, someexamples being the NO and arylhydrocarbon receptors.

2.3. Measurementof Receptor-Ligand Interaction

Hormone receptors are most commonly detected andmeasured using direct binding assays. This requires alabeled ligand, usually a radiolabeled hormone such asa tritiated steroid or an iodinated peptide; a source of the

receptor being analyzed, typically tissue extracts, cells,or cell membranes; and a physical means of separatingthe bound hormone from that which is unbound, com-monly centrifugation or filtration techniques. As shownin Fig. 2A, when increasing amounts of a labeledpolypeptide hormone are added to a constant amount ofa cell preparation, the fraction of hormone bound to thecell surface increases and eventually approaches a maxi-mum. In addition to specific binding to its cell surfacereceptor, the hormone can bind nonspecifically to othercellular constituents or to the reaction vessel, and thiscomponent is commonly measured by performing thebinding reaction in the presence of a large excess ofunlabeled hormone, to ensure complete displacement ofthe labeled hormone from specific sites. Specific bind-ing is then established by subtracting the nonspecificbinding from the total binding.

In a useful variation of this assay, a competition bind-ing experiment, a constant, small amount of the labeledhormone is mixed with cells or cell membranes in thepresence of increasing concentrations of unlabeled hor-mone. A typical competition or displacement curve re-

Fig. 2. Measurement of hormone-receptor interactions by binding analysis: (A) saturable binding to a target cell preparation asconcentration of radiolabeled hormone is increased; (B) competition or displacement experiment in which binding of a radiolabeledtracer to the receptor is competed as excess unlabeled hormone is added; (C) Scatchard linear transformation of binding data, fromwhich receptor number and affinity for hormone can be determined; (D) consequence of spare receptors. The biologic response isfully activated when only a fraction of the receptors is occupied by hormone. See text for details.


sulting from this type of experiment is shown in Fig. 2B.The binding that remains at high hormone concentra-tions represents the nonspecific binding, and the hor-mone concentration at which 50% of the radiolabeledtracer is displaced approximates the affinity constant ofthe receptor for its ligand, as described next.

The interaction of the hormone and its receptor canbe described by the equation H + R� [HR], in which His the free hormone, R is the free receptor, and HR is thehormone-receptor complex. Binding is assumed to bereversible, and the steady-state dissociation constant Kd

= [H][R]/[HR] provides a measure of the affinity of thereceptor for its ligand. The total amount of receptor canbe described as that which is free plus that which isliganded ([RT] = [R] + [HR]), and the two equationstogether can be written in the form [HR] = [RT][H]/[H]+ Kd. When [HR] is plotted as a function of [H], thehyperbolic plot shown in Fig. 2A is obtained. The pre-vious equation can be rearranged to the form [HR]/[H]= [RT]/[H] + Kd, and when [HR]/[H] is plotted as a func-tion of log[H], the sigmoidal curve shown in Fig. 2B isobtained. Scatchard (1949) described a commonly usedlinear rearrangement of this relationship, [HR]/[H] =–1/Kd [HR] + RT/Kd, and as shown in Fig. 2C, the plotof [HR]/[H] as a function of [HR] provides the totalreceptor number, RT as the intercept with the abscissa,and the affinity constant, Kd, is derived from the slope,which is –1/Kd. The linear relationship shown in Fig. 2Cbecomes curvilinear when multiple binding sites withdiffering affinities are present or when cooperative bind-ing interactions occur.

Most commonly, the Kd values for hormone recep-tors are near the physiologic concentration of the rel-evant hormone, meaning that fluctuations in thehormone level can elicit both positive and negativeresponses with respect to receptor activation. How-ever, in many cases it has been shown experimentallythat occupancy of only a small fraction of the cell-surface receptors is needed to elicit a maximal bio-logic response. The term spare receptors has evolvedto describe this phenomenon, which is illustrated interms of binding and activation curves in Fig. 2D. Theability of a small fraction of occupied receptors tostimulate fully a biologic response points to the tre-mendous amplification potential of the enzyme effec-tors that act downstream of the hormone receptors.Spare receptors may allow a response to be kineticallyfavorable even at very low hormone concentrations.

2.4. Cellular Signal Transduction ThemesHormones, particularly those that act on cell-surface

receptors, most commonly initiate a cascade of signal-ing events in their target cell, ultimately leading to an

appropriate biologic response. These cascades arereferred to as signal transduction pathways, and althoughthey are extremely diverse with respect to the specificmolecules involved, they do have several common fea-tures. It is becoming clear that in many systems, homo-or heterodimerization of receptor proteins in responseto binding of the hormone ligand is a key event in ini-tiating cell signaling. This can lead to direct activationof a receptor’s enzymatic function, or to the direct orindirect recruitment of additional effector proteins withenzymatic function. Through the actions of soluble sec-ond-messenger molecules, or through the direct enzy-matic functions of the receptor or its associated proteins,protein kinases, protein phosphatases, or proteases areoften activated and act to initiate cascades of proteinmodification that can be extraordinarily complex. Someof the modified target proteins play direct and rapidroles in the cellular response (e.g., an ion channel)whereas other modified target proteins play indirect ordelayed roles in the cellular response (e.g., a transcrip-tion factor). Another common feature is the presence ofregulatory mechanisms that attenuate the response tohormone and reset the signaling system. Chapter 3 pro-vides a comprehensive treatment of signal transduction,and, therefore, only a few key points necessary for thesubsequent discussion of hormone receptors and theiractions are introduced in this section.

One can consider three broad classes of signal trans-duction themes downstream of cell-surface receptors:activation of second-messenger signaling pathways byG protein–coupled receptors (GPCRs), activation oftyrosine kinase or serine/threonine kinase receptors orreceptor-associated proteins by growth factor andcytokine receptors, and activation of protein proteoly-sis or cell localization pathways by developmentallyimportant receptors. Each of these is very briefly reviewedhere and is considered in more detail in the followingsections.

GPCRs are the largest single family of receptors invertebrates, and derive their name from their commonmechanism of action, which is to activate one or moreG proteins following hormone stimulation. The G pro-teins are a large family of guanosine 5´-triphosphate(GTP)-binding proteins that are heterotrimeric, consist-ing of α-, β- and γ-subunits, and serve to stimulate orrepress the activity of multiple cellular effector enzymes(Gilman, 1987). Some 16 distinct G protein α-subunitsdefine major categories of cell responses; for example,stimulatory (Gs) and inhibitory (Gi) G proteins activateor repress, respectively, the enzyme adenylyl cyclaseand thereby regulate production of the second-messen-ger cAMP (Schramm and Selinger, 1984), and Gq andG11 class G proteins regulate phopholipase Cβ activity


and control the production of inositol phosphate, diacyl-glycerol, and calcium second messengers (Berridge,1993). Both the α and βγ G protein subunits regulateenzyme effectors, and a cycle of GTP binding and sub-sequent hydrolysis to guanosine 5´-diphosphate (GDP)controls the subunit association and activity of theseproteins. The second messengers that are produced as aresult of G protein activation serve to regulate the activ-ity of cellular protein kinases, such as the activation ofcAMP-dependent protein kinase (protein kinase A[PKA]) and PKC by cAMP and calcium/phospholipids,respectively.

A second common signaling theme involves cell-surface receptors that have intrinsic protein kinaseactivity or that recruit and activate protein kinases.Three subclasses of these receptors can be considered.A large and historically important group is the growthfactor receptors, which have cytoplasmic domains withintrinsic protein tyrosine kinase activity. On hormonebinding, the receptor rapidly forms a dimer, and thecytoplasmic kinase domains rapidly cross-phosphory-late each other on tyrosine residues, which allows forthe subsequent binding of a broad repertoire of cellularproteins that are involved in transducing the signal. Amore recently characterized group is the transforminggrowth factor-β (TGF-β) superfamily receptors, whichhave cytoplasmic domains with intrinsic protein serine/threonine kinase activity. Dimerization of a hormone-binding type II receptor and a signaling type I receptorleads to the phosphorylation of target proteins of theSmad family, which transduce the signal. Finally, thecytokine receptors are not themselves protein kinases,but when activated they recruit soluble protein tyrosinekinases of the Janus kinase (Jak) family that phospho-rylate both the receptor and receptor-associated effec-tor proteins of the Stat family, which transduce thesignal. Also in the same broad category are receptorsthat are protein tyrosine phosphatases, although muchless is known about these receptors and their activation.

A third broad and emerging category includes path-ways downstream of a variety of developmentally im-portant signaling molecules such as the Wnt, Hedgehog,and DSL family ligands. The details are quite specific toeach pathway, but common themes include retention ofa key transcription factor or coregulator required fortarget gene expression in the cytoplasm until ligandactivates the receptor, a switch from target gene repres-sion to activation, and involvement of irreversible pro-teolysis as a key regulatory step.

2.5. Mechanisms of Receptor RegulationIt is clear that mechanisms must exist to regulate the

levels of, or activity of, cell-surface receptors, allowing

for modulation or termination of the response to thehormone. In some cases, the biosynthesis of receptors istightly regulated so as to generate additional receptorswhen they are required. For example, the low-densitylipoprotein (LDL) receptor gene is activated by a pro-tein that acts as both a sterol sensor and transcriptionfactor, resulting in enhanced production of the receptorwhen sterol levels are low (Brown and Goldstein, 1999).In other cases, the degradation of receptors, and theircorresponding ligands, is tightly regulated. Many hor-mones that act on cell-surface receptors are internalizedthrough the process of receptor-mediated endocytosis,which is shown in Fig. 3A. The hormone-receptor com-plex is internalized into clathrin-coated vesicles that areacidified and lose their clathrin coat to form endosomes,where the low pH often results in hormone-receptor dis-sociation. The ligand is most commonly degraded inlysosomes, effectively removing the signal from theextracellular environment. The receptor typically hasone of two fates: recycling to the cell surface, where itis again available to interact productively with hormone(e.g., the insulin receptor); or degradation in the lyso-some (e.g., the epidermal growth factor receptor[EGFR]). In either case, internalization effectivelyreduces the number of cell-surface receptors, and theprocess is therefore a mechanism utilized to down-regulate cell-surface receptors.

A second common mode of regulation targets the abil-ity of cell-surface receptors to bind the hormone or tosubsequently transduce a signal. Probably the best-stud-ied example of this is agonist-induced desensitization ofthe G protein–coupled β-adrenergic receptor. In the con-tinual presence of the agonist epinephrine, the cAMPsignal is attenuated, although the receptor continues tobind epinephrine. Desensitization is a reversible pro-cess, and it is mediated by phosphorylation of the recep-tor on cytoplasmic serine and threonine residues. Someof this phosphorylation is catalyzed by PKA, which isactivated in response to the epinephrine-induced cAMPsignal and acts in a feedback manner to downregulatethe signaling pathway. Because activated PKA has theability to phosphorylate many receptors, potentiallyattenuating their activity, this phenomenon is referred toas heterologous desensitization. The agonist-occupiedβ-adrenergic receptor is also phosphorylated by a veryspecific cellular kinase called the β-adrenergic receptorkinase (βARK), a member of a broader family of GPCRkinases, or GRKs (Claing et al. 2002). These kinases arebroadly expressed and likely to play an important role inthe desensitization of many hormonal responses medi-ated by GPCRs. Because βARK targets only the β-adr-energic receptor, this phenomenon is referred to ashomologous desensitization. The phosphorylated β-adr-


Fig. 3. Mechanisms for regulation of cell-surface receptors. (A) Process of receptor-mediated endocytosis. Following ligand binding,receptors rapidly move into coated pits and are internalized by endocytosis. The resulting clathrin-coated vesicles are acidified inan endosomal compartment, leading to dissociation of the ligand from the receptor. Vesicular sorting leads to a segregation of theligand into vesicles that will fuse with primary lysosomes, resulting in degradation or utilization of the ligand. The receptor eithercan be degraded, as often occurs for signaling receptors, or it can be recycled to the cell surface, as often occurs for transport receptors.(B) Steps involved in desensitization of β-adrenergic receptor. In the continual presence of hormone, the G protein βγ-subunits recruitthe β-adrenergic receptor kinase to the membrane, leading to specific phosphorylation of the receptor and an attenuation of signaling.The phosphorylated receptor is recognized by β-arrestin, further suppressing the signaling pathway. The pathway shown is homolo-gous desensitization. As discussed in the text, heterologous desensitization involving receptor phosphorylation by PKA also occurs.ATP = adenosine triphosphate.

energic receptor is unable to couple efficiently to its Gprotein, resulting in the observed attenuation of the sig-nal. In addition, many phosphorylated GPCRs, includ-ing the β-adrenergic receptor, bind proteins of thearrestin family, which serve to suppress further the sig-nal by promoting uncoupling of the receptor and its Gprotein (Claing et al., 2002). Some of the regulatoryprocesses involved in receptor desensitization are out-lined in Fig. 3B. Desensitization is a major factor con-trolling the efficacy of action of many therapeutic agentsthat target GPCRs such as the β-adrenergic receptor.Although negative regulatory mechanisms for otherclasses of receptors have not been studied in the samedepth as those for the GPCRs, they certainly exist, andseveral are referred to in subsequent sections.

3. CELL-SURFACE RECEPTORS3.1. Receptors Coupled to G Proteins

The GPCRs comprise the largest known family ofcell-surface hormone receptors, and it is estimated that50% of prescription drugs target GPCRs. Hundreds ofthese receptors have already been cloned and charac-terized, many of them orphan receptors of unknownfunction that represent significant targets for futuredrug development. Their common features are sevenhydrophobic potential membrane-spanning domains,and the ability to stimulate the exchange of bound GDPfor GTP on associated G protein α-subunits in responseto agonist binding. They are also known as the hepta-helical or serpentine receptors because of their mem-


16

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brane topology. As indicated in Fig. 4A, the GPCRsuperfamily can be divided into several major familiesthat share significant sequence similarities. The threepredominant mammalian families are class A (recep-tors related to rhodopsin and the β2-adrenergic recep-tor), class B (receptors related to the secretin,calcitonin, and parathyroid hormone [PTH] receptors),and class C (receptors related to the metabotropicglutamate and pheromone receptors). Further subdivi-sions into groups are indicated in Fig. 4, as are receptorclasses D–E. Class A is by far the largest family ofGPCRs, and its prototypes, rhodopsin and the β2-adrenergic receptor, are among the best-characterizedreceptors; therefore, much of what is known about thestructure, function, and regulation of GPCRs in gen-eral comes from studies of these model proteins.

Tremendous diversity is a hallmark of the GPCRsuperfamily. Many of the neurotransmitter and peptidehormone receptors are encoded by multiple genes thatproduce related yet distinct receptors. For example, fivedistinct somatostatin receptors have been characterized.These are expressed with unique but overlapping tissueand cell specificity, and they are able to mediate signal-ing through several different G proteins. An extremeexample of this is represented by the olfactory odorantreceptors, a family that likely numbers in the hundreds.Other receptors are produced from a single gene, butalternative RNA processing results in the generation ofmultiple receptor isoforms, either produced in specifictissues or able to couple differentially to G protein–mediated signaling pathways. For example, the dopam-ine D2 receptor exists as two splice variants expressedin unique tissue-specific patterns, and the pituitary ade-nylate cyclase activating peptide receptor exists as fivesplice variants that differ in their ability to activate ade-nylate cyclase vs phospholipase Cβ (PLCβ) effectors.Further diversity may be generated by recent findingsthat both homo- and heterooligomerization of GPCRs iscommon and can impact function, potentially generat-ing receptors with novel properties through interactionof characterized receptors with known properties(George et al., 2002).

The major structural features of a model GPCR, theβ2-adrenergic receptor, are shown in Fig. 4B. The ex-tracellular domain is relatively short, although for someclass A receptors, those for the glycoprotein hormonesFSH, luteinizing hormone, and thyroid-stimulating hor-mone, this domain is more than 300 amino acids long.There are typically several sites for asparagine-linkedglycosylation within the extracellular domain. Theseven membrane-spanning domains of the receptor cre-ate three extracellular loops and three cytoplasmic loopsthat can be quite variable in length among different

receptors. The carboxyl-terminal cytoplasmic domainis typically short and is often associated with the plasmamembrane through palmitoylation of a conserved cys-teine residue. As discussed above, phosphorylationplays an important role in the regulation of receptoractivity, and potential sites for phosphorylation by PKAas well as the specific GRK kinases are found within thethird cytoplasmic loop and the carboxyl-terminal tail ofthe receptor.

Mutagenesis has been extensively applied to eluci-date features of the β2-adrenergic receptor important forligand interaction and for subsequent G protein cou-pling. A strategy that has been particularly useful for theanalysis of the GPCRs has been the generation of chi-meras between two homologous receptors with distinctligand-binding or signaling properties. Such chimerasbetween the β2-adrenergic and α1-adrenergic receptorshave implicated the transmembrane domains and asso-ciated extracellular loops in specific agonist binding andhave shown that transmembrane domain seven is par-ticularly important in specifying antagonist binding.Similar experiments to examine signaling through thestimulation (β2-adrenergic receptor) or inhibition (α1-adrenergic receptor) of adenylate cyclase by Gs and Gi,respectively, led to the conclusion that the regions be-tween transmembrane domains five and six, includingthe third cytoplasmic loop, were particularly importantin determining G protein recognition. Additional muta-genesis has indicated that the carboxyl-terminal domainof the receptor, a major site of potential phosphoryla-tion, is particularly important for desensitization, andthat several conserved residues are critical for cata-cholamine binding and activation, including an asparticacid residue in transmembrane domain two and twoserine residues in transmembrane domain five of the β2-adrenergic receptor (Dohlman et al., 1991).

In general, much of what has been learned from modelreceptors such as the β2-adrenergic receptor has beenapplicable to additional GPCRs. The basic membranetopology of the β-adrenergic receptor, discerned throughproteolysis and antibody epitope mapping studies, holdsfor other members of the superfamily that have beenexamined. There are differences in ligand-bindingdeterminants, and, in general, receptors that interact withsmall molecules utilize the transmembrane domain seg-ments more extensively for binding, and receptors thatinteract with larger peptides or proteins make greateruse of the amino-terminal and extracellular loops. Anextreme example of this is ligand recognition by theglycoprotein hormone receptors, where the large amino-terminal extracellular domain, expressed independentlyof the transmembrane domains, has the ability to bindthe ligand with high affinity. As additional members of


this family are characterized, novel modes of hormonebinding and receptor activation are also being observed.For example, thrombin proteolytically cleaves itsGPCR in the extracellular domain, revealing a new N-terminus that acts like a ligand to mediate activation ofthe receptor.

The mechanisms by which ligand binding leads toan ability of the receptor to interact productively witha G protein remain largely unknown. The first (and todate only) structure for a GPCR is that of rhodopsin(reviewed in Filipek et al., 2003), and this has providedan important foundation for modeling studies of otherGPCRs. Based on a wealth of biophysical and mutagen-esis data, researchers believe that activation of GPCRsinvolves disruption of stabilizing contacts betweenmembrane-spanning helices, causing changes in therelative orientation of several of these helices; in par-ticular, there is a movement of transmembrane helix 6with respect to transmembrane helix 3, opening up thereceptor in a manner that is thought to expose cyto-plasmic determinants for G protein interaction. Asdiscussed in Section 5, mutations in GPCRs that causeconstitutive activity of the receptor are a commoncause of disease. In addition, many GPCRs are par-tially active in the absence of ligand and can be furtheractivated when overexpressed. This has been explainedin terms of a two-state model in which an equilibriumexists between a signaling active and inactive state ofthe receptor. Agonists bind to and stabilize the activeconformation, whereas inverse agonists bind selec-tively to the inactive conformation and stabilize it; thislatter class of compounds is a potential target for drugdevelopment in regulating the activity of constitutivelyactive mutant receptors (Strange, 2002).

As discussed earlier, the functional commonalityamong GPCRs is that following hormone binding theyinteract with the G protein so as to induce the release ofGDP from the G protein α-subunit, allowing GTP tobind and facilitating dissociation of the α-subunit fromthe βγ complex. Several G protein structures have beensolved using crystallography, and these provide sub-stantial molecular detail into how guanine nucleotidebinding impacts subunit interactions. Both the free α-subunit and the βγ complex participate in signaling,activating one or more effector enzymes. Eventually,the intrinsic guanosine 5´-triphosphatase (GTPase)activity of the α-subunit hydrolyzes GTP to GDP, pro-moting reassociation with the βγ complex and return tothe inactive state. For some G proteins with slow intrin-sic GTPase activity, this process is facilitated byGTPase-activating proteins called regulators of G pro-tein signaling. Aspects of signaling by GPCRs are sum-marized in Fig. 4C, which illustrates the best-studied

signaling pathway activated by this class of receptors,the Gs-stimulated cAMP pathway.

Given the diversity of the GPCRs, it is perhaps notsurprising that many endocrinopathies can be attrib-uted to mutations in specific GPCRs (Shenker, 1995).This connection first became apparent through theanalysis of G proteins that these receptors interact with.It was demonstrated that mutations that constitutivelyactivate Gsα by inactivating its intrinsic GTPase func-tion were causative in acromegaly in association withpituitary adenoma (a late somatic mutation confined tothe pituitary gland) as well as in McCune-Albright dis-ease (an earlier somatic mutation affecting many endo-crine systems). Subsequently, mutations in manyGPCRs have been identified, both in human diseasesand in animal models of human disease. These representboth loss-of-function and gain-of-function mutations inthe targeted GPCRs. For example, inactivating muta-tions of the calcium-sensing receptor cause familialhypocalciuric hypercalcemia in the heterozygous stateand neonatal severe hyperparathyroidism in the homo-zygous state, whereas an activating mutation in this samereceptor causes a dominant form of hypocalcemia. Insome cases, analysis of these mutations, which gener-ally have a known phenotype, has revealed much aboutimportant structural features of these receptors. Forexample, more than 50 different mutations of the vaso-pressin V2 receptor in patients with nephrogenic diabe-tes insipidus have been found, providing a wealth ofinformation on residues essential for hormone bindingor activation of this receptor.

3.2. Receptors With Tyrosine Kinase ActivityThe receptor protein tyrosine kinases (RTKs) are

found in all multicellular organisms and mediate theactions of a broad spectrum of hormones and growthfactors on cell growth, metabolism, and differentia-tion. They can be subdivided into several families,based largely on their structural characteristics. Exam-ples of some of the major subfamilies, by one classifi-cation (van der Geer et al., 1994), include the platelet-derived growth factor receptor (PDGFR) subfamily,the fibroblast growth factor receptor (FGFR) subfam-ily, the insulin receptor subfamily, the EGFR sub-family, the nerve growth factor receptor (NGFR) sub-family, the hepatocyte growth factor receptor (HGFR)subfamily, and a series of additional receptors forwhich ligands have not yet been identified. Figure 5Ashows examples of the major families of RTKs.

The RTKs are type I transmembrane proteins, havingan external aminoterminus and a single membrane-span-ning domain. The extracellular ligand-binding domains(LBDs), of these receptors are quite distinctive, but one


or more of a variety of recognizable structural motifsappear in most of the LBDs. These domains includecysteine-rich regions, leucine-rich regions, immunoglo-bulin-related domains, fibronectin type II repeats, andEGF-like repeats, among others (Fig. 5B). For the mostpart, the precise roles of these extracellular domains inhormone recognition have not been well established,but structural studies are beginning to shed new light onthe processes of hormone binding. The cytoplasmiccatalytic domain of the RTKs is much more highly con-served, and a number of amino acid residues critical toits enzymatic function are absolutely conserved. Basedon the crystallographic structures of several solubleprotein kinases as well as the insulin receptor and EGFRtyrosine kinase domains, it appears that the receptorprotein tyrosine kinase catalytic domains are likely toconsist of two lobes, one that binds Mg2+/adenosinetriphosphate (ATP) utilizing a GXGXXG motif, andone that forms the actual catalytic loop. In some recep-tors in this family, such as the PDGFR, the tyrosine

kinase catalytic domain is interrupted by a spacer region(Fig. 5B).

The RTKs all exhibit a hormone-dependent dimer-ization that is crucial for subsequent signal transduc-tion. Crystallographic studies of the extracellulardomains of the EGFR and its relatives ErbB2 (Neu/HER2) and ErbB3 (HER3) demonstrate that growthfactor binding to each receptor monomer promotes astructural rearrangement to expose a dimerization inter-face. For ErbB2, which does not have a ligand, thedimerization domain is constitutively exposed (Burgesset al., 2003). Some ligands for these receptors are them-selves dimers (e.g., PDGF), whereas others are mono-mers (e.g., EGF), and this may affect the dimerizationmechanism. The functional consequence of receptordimerization is to bring the cytoplasmic tyrosine kinasedomains into close proximity, which leads to cross-phosphorylation of the receptors and the subsequentrecruitment of substrates having affinity for the tyrosine-phosphorylated receptor. These include a broad reper-

Fig. 5. Structure and signal transduction by RTK: (A) major families and specific examples of RTKs; (B) schematic of structures ofseveral types of RTKs and key modular domains involved in ligand interaction; (C) example of generic signaling through a prototypicRTK, leading to activation of a MAPK signaling pathway as exemplified by ERK kinase.


toire of cellular proteins that have an Src homology 2(SH2) domain that specifically recognizes tyrosinephosphorylation sites on the receptor. Some of theseSH2 domain proteins are direct substrates for phospho-rylation by the receptor and can serve as effector mol-ecules in transducing the signal. Examples of substratesinclude the phosphatidylinositol-3-kinase regulatorysubunit, the Src family protein kinases, the protein tyro-sine phosphatase Syp, and PLCγ. Other SH2 domainproteins act simply as molecular adapters; they bind tothe phosphorylated receptor and also associate withadditional cellular proteins, commonly through a pro-tein interaction motif referred to as the SH3 domain.Examples include Grb2, Nck, and Crk, which bind to avariety of hormone receptors. Several proteins, includ-ing the adapter Shc and the insulin receptor substratefamily of docking proteins, utilize a distinct phos-photyrosine-binding domain that recognize an Asn-Pro-x-pTyr motif in target receptors. Yet other adapter andeffector proteins in this signaling pathway containpleckstrin homology domains, which may play a role intethering these proteins at the plasma membrane, orcontain WW protein interaction motifs. Use of modularinteraction domains is a common theme in cell signalingand is particularly apparent in the RTKs (Pawson andScott, 1997).

Adapter proteins mediate a broad spectrum of cellu-lar responses, but a common and well-characterizedresponse leads to activation of the small GTP-bindingprotein Ras. Ras activation initiates a kinase cascade,the mitogen-activated protein kinase (MAPK) cascade,which eventually results in the phosphorylation andactivation of nuclear transcription factors able to altergene expression in the target cell. At many of the keyregulatory steps, specific protein tyrosine phospha-tases counter the actions of the kinases and, thus, exertnegative control over activation, a likely component ofinhibitory feedback mechanisms. A generic example ofsignaling of an RTK through the MAPK pathway lead-ing to extracellular-regulated kinase (ERK) activationis shown in Fig. 5C.

The physiologic functions of many of the receptors inthe tyrosine protein kinase family have been exploredthrough gene disruption approaches in mice, or by theidentification of naturally occurring mutations in thesereceptors in animal models or in people. It should berealized that many of the RTKs were initially discov-ered as transduced retroviral oncogenes that representmutated, truncated, or inappropriately expressed ver-sions of their cellular counterparts (Carbone and Levine,1990). Although a complete discussion of the physi-ologic functions of receptor tyrosine protein kinases isbeyond the scope of this chapter, a few examples related

to several important model receptors (the EGF, PDGF,and insulin receptors) are briefly considered. The EGFRplayed a key role in defining the relationship of cellularsignaling pathways to retrovirus-induced cellular trans-formation and oncogenesis. A truncated and trans-duced form of the EGFR represents one of two onco-genes (v-erbB) of the avian erythroblastosis virus, andthe highly related Neu tyrosine kinase receptor (alsoreferred to as HER2 or erbB2) is mutated or amplifiedin a wide variety of human cancers. Thus, inappropriateactivation of the signaling pathways mediated by thesetyrosine kinase receptors can lead to cell transformationand tumorigenesis. Consistent with this, the PDGFR isinvolved in a number of reciprocal chromosomal trans-locations that lead to its constitutive activation and areassociated with myeloproliferative disorders. The insu-lin receptor is subject to mutation in patients with insu-lin resistance, and this has led to the identification ofmany different types of mutations that affect the synthe-sis or function of this RTK. These mutations are associ-ated with type A insulin resistance, leprachaunism, andlipoatrophic diabetes.

3.3. Receptors With Serineand Threonine Kinase Activity

Receptors with serine and threonine kinase activitywere identified relatively recently, beginning with theexpression cloning of the activin type II receptor in 1991(Mathews, 1994). Activin is a protein hormone in theTGF-β superfamily, and this superfamily includes hor-mones such as Müllerian-inhibiting substance (MIS),the bone morphogenetic proteins (BMPs), and nonmam-malian homologs such as Drosophila decapentaplegic(dpp) and Xenopus Vg-1. Receptor nomenclature hasbeen based on early crosslinking studies with TGF-βthat revealed three distinct protein bands designated typeI, II, and III receptors. The type III receptor is a largeproteoglycan, also known as beta glycan, which isthought to play a role as a coreceptor for TGF-β, inhibin,and possibly other family members by facilitating deliv-ery of the hormone to the signaling receptors. Both thetype I and II receptors are involved in signaling. Distinctcombinations of type I and II proteins form the func-tional receptor for each ligand of the TGF-β superfam-ily, and the relative roles of the two receptor subunitsvary somewhat, depending on the ligand with whichthey interact. Figure 6A shows several representativeligands of the TGF-β superfamily along with the type Iand II receptors that mediate their actions.

Both the type I and II receptors have conserved cyto-plasmic domains with protein serine or threonine kinaseactivity. For those ligands most closely related to TGF-β, including activin and nodal, the type II receptor is the


high-affinity ligand-binding determinant. Binding ofhormone allows the type II receptor to interact with thetype I receptor, most likely in the form of a heterotetra-mer that interacts with the homodimeric ligand (Fig. 6B).The type II receptor, which has constitutive kinaseactivity, then phosphorylates the type II receptor in aserine-rich juxtamembrane region known as the GS box,leading to activation of the type I receptor kinase. Bycontrast, for ligands related to the BMPs, including thegrowth and differentiation factors (GDFs) and MIS, it isthe type I receptor that has a higher affinity for ligand,although the type II receptor does contribute to ligandbinding (Fig. 6B). Oligomerization leads to phosphory-lation and activation of the type I receptor. Substantialinsight into the interactions between ligand and receptorin this system has emerged from crystallographic andnuclear magnetic resonance structural studies (Shiand Massague, 2003). There appears to be a well-con-served mechanism for binding of BMP-related ligandsto the type I receptor, but there is substantial diversity inthe way in which TGF-β-related ligands interact withthe type II receptor.

The major transducers of signaling downstream ofthe serine and threonine kinase type I receptors are afamily of proteins known as the Smads in vertebrates.The name derives from the related proteins in Caenor-habditis elegans (Sma) and Drosophila (Mad). Thereare eight known Smad proteins that play distinct roles insignaling between the cell-surface receptor and thenucleus. Smads 1, 2, 3, 5, and 8 are known as receptor-regulated Smads (R-Smads), and they are direct targetsfor phosphorylation within a C-terminal SSXS motif bythe type I receptor. The TGF-β-related ligands lead tophosphorylation of Smads 2 and 3, and the BMP-relatedligands lead to phosphorylation of Smads 1, 5, and 8.The phosphorylated Smad proteins dissociate from thereceptor and form heteromeric complexes with a com-mon cytoplasmic Smad protein, Smad4. This hetero-meric complex moves to the nucleus and acts to modu-late transcription of target genes. Although there is evi-dence for direct DNA-binding by Smads 3 and 4, thepredominant actions of Smads are thought to be medi-ated by their interaction with nuclear DNA binding pro-teins and stabilization of ternary DNA-bound complexes

Fig. 6. Structure and signal transduction by receptor serine/threonine kinases: (A) representative ligands in TGF-β superfamily, theirtype II and I receptors, and Smad proteins they activate; (B) mechanisms of ligand binding to receptors for the two major subclassesof TGF-β ligands, TGF-β class and BMP class; (C) example of generic signaling through the Smad pathway, although this exampleof ligand binding illustrates initial binding to type II receptor, which is characteristic of TGF-β class.


among the R-Smads, Smad-4, and the target transcrip-tion factor (Attisano and Wrana, 2000). The Xenopustranscription factor FAST-1 was the first of these inter-acting proteins to be identified, but it is likely that theSmads target many transcriptional regulatory proteins.Smads 6 and 7 are known as inhibitory Smads, and theyact to inhibit the activation of the R-Smads by bindingto the type I receptor. Smads 6 and 7 are up-regulated byBMP and TGF-β pathway signaling, respectively, andare likely part of a negative feedback system. Finally,there are numerous examples of additional proteins thatinteract with or modify the activity of the serine andthreonine kinase receptors, and signaling crosstalk islikely a very important component in generating speci-ficity of response. Figure 6C summarizes aspects of sig-naling downstream of serine and threonine kinasereceptors.

There are several reports of mutations in these recep-tors in association with human disease. Mutations in theBMP type II receptor and in ALK1 are found in pulmo-nary hypertension, and mutations in ALK1 are alsofound in hereditary hemorrhagic telangiectasia type II.Loss of expression of the TGF-β type II receptorthrough microsatellite instability or as a result of tran-scriptional repression is found in a variety of humancancers. Much more frequent are mutations in the down-stream Smad proteins, indicating a role for these pro-teins as tumor suppressors. Smad2 mutations are foundin colorectal and lung cancers, and Smad4 mutationsare found in many cancers but are particularly prevalentin pancreatic carcinoma, in which Smad4 was first iden-tified as the tumor suppressor deleted in pancreaticcancer (dpc)-4.

3.4. Receptors That Recruit Tyrosine KinasesMany well-characterized cell-surface receptors lack

apparent enzymatic functions within their cytoplas-mic regions, and their activity therefore depends on therecruitment of signaling proteins to the receptor. Thisfamily includes the receptors for the interleukins (ILs),interferons (IFNs), erythropoietin (EPO), granulocyte-macrophage colony-stimulating factor (GM-CSF), leu-kemia inhibitory factor (LIF), growth hormone (GH)and prolactin (PRL), among others, and is known as thecytokine receptor superfamily (Fig. 7A). The functionalreceptors include single-chain type I transmembraneproteins (GH, PRL, EPO), those that have a ligand-spe-cific subunit that interacts with a common shared sub-unit (ILs, GM-CSF, LIF) and those that have multipleunique subunits (IFNs). Some examples of cytokinefamily receptors of each type are shown in Fig. 7B.Although these receptors all lack tyrosine kinase activ-ity, conserved membrane-proximal regions of their

cytoplasmic domains mediate ligand-dependent inter-action with one or more cytoplasmic protein tyrosinekinases, the Janus kinases, or Jaks (Ihle and Kerr, 1995).These kinases, including Jak1, Jak2, Jak3, and Tyk2,have amino-terminal homology domains of unknownfunction, and two carboxyl-terminal kinase domains,although the first of these lacks critical residues neces-sary for catalytic activity and is inactive. The Jak kinasesare tyrosine phosphorylated and activated followingligand binding to the cytokine receptors, and this requiresan association of the Jak kinases with two conservedmembrane-proximal motifs in the receptor referred toas box 1 and box 2. It is thought that receptor dimeriza-tion brings the associated Jak kinases into close proxim-ity and allows cross-phosphorylation and activation tooccur, and, thus, two Jak kinases associate in the samemanner as two cytoplasmic kinase domains of the RTKsassociate. In addition to Jak kinase phosphorylation, theactivation process is associated with phosphorylation ofthe receptor itself on specific tyrosine residues, whichcan lead to activation of some of the same pathwaysactivated by the conventional RTKs, such as the MAPKpathway.

The pathway from Jak kinase activation to changesin target cell gene expression was completed throughthe analysis of proteins mediating the transcriptionalresponses to IFN, a family of proteins known as theStat (signal transducer and activator of transcription)proteins (Darnell et al., 1994). Following cytokinereceptor phosphorylation by a Jak family kinase, Statproteins are recruited to the receptor and phosphory-lated on a single tyrosine residue near the C-terminaltransactivation domain of the protein. The phosphory-lated and activated Stat proteins form homo- orheterodimers, translocate to the nucleus, and bind tospecific DNA elements (originally called γ IFN activa-tion sequences, or GAS elements) near target genes toregulate transcription. Stat proteins include a highlyconserved SH2 domain as well as an SH3-relateddomain in the C-terminal regions, in addition to theconserved C-terminal tyrosine phosphorylation site,and are reported to interact with a broad spectrum ofother transcription factors and coregulators. Seven Statproteins that fall into three groups have been identi-fied, and it appears that these serve as the major linkbetween cytokine receptor activation and cellular tran-scriptional responses. There are likely several modesof negative regulation of this pathway, but a majormechanism involves Stat protein induction of the sup-pressor of cytokine signaling (SOCS) family of pro-teins, which then act through mechanisms that remainunclear to block Stat signaling, most likely at the levelof the receptor or Jak kinase (Cooney, 2002).


It is clear that different receptors in the cytokine re-ceptor superfamily preferentially associate with one ormore of the four Jak kinases and subsequently preferen-tially activate one or more of the seven Stat proteins, butthe mechanisms of specificity are largely unknown. Thevarious Jaks and Stats have unique tissue distributions,and this likely explains in part why particular receptorssignal through particular Jak-Stat pathways. Becausethe cytokine receptor superfamily is large, and the recep-tors utilize diverse signaling mechanisms, it is informa-tive to consider one example focused on the endocrinesystem in greater detail. One of the best-studied recep-tors in this class is the GHR. It is representative of thesingle-chain class of cytokine receptor and has the twopairs of conserved cysteines in the extracellular domainthat are a hallmark of this family. Interestingly, tworeceptor chains bind to a single GH molecule at distinctsites on the hormone, and thus a single ligand moleculecatalyzes receptor dimerization and subsequent signal

transduction. This has been demonstrated by deter-mination of the crystallographic structure of the extra-cellular domain of the GHR complexed with GH. Site 1on GH must be occupied prior to occupancy of site 2.At high concentrations of hormone, saturation of allreceptors by binding to site 1 on GH is predicted toinhibit receptor dimerization and the subsequent bio-logic response. This has been experimentally demon-strated in several systems and may represent anadditional mode of receptor regulation. The GHR isunique in that the extracellular domain of the receptoralso functions as a soluble serum-binding protein forGH. The extracellular domain is released from themature receptor by proteolysis in some species, and it isencoded by a distinct mRNA generated by alternativeRNA processing in other species. The activated GHRassociates with the Jak2 kinase, leading to the phospho-rylation and activation of Stats 1, 3, and 5. In addition,the MAPK pathway is also activated by GH, presum-

Fig. 7. Structure and signal transduction by cytokine superfamily receptors: (A) examples of some of the major families of receptors,along with Janus family kinases and Stat proteins they activate; (B) representative schematic structures demonstrating subunitcomposition of various types of cytokine superfamily receptors; (C) example of generic signaling through a type I cytokine receptorto the Jak-Stat pathway.


ably through the interaction of adapter proteins with thephosphorylated GHR, or with Jak2 itself. Signalingthrough a single-chain cytokine superfamily receptorsuch as the GHR is illustrated in Fig. 7C.

The GHR is also a good example of the potentialinvolvement of receptors within this superfamily inendocrine disease. Mutations in the GHR/binding pro-tein are responsible for the GH resistance observed inLaron-type dwarfism, in which serum GH is elevated inassociation with classic symptoms of GH deficiency,including reduced levels of insulin-like growth factor-1 (IGF-1) and short stature. Dozens of distinct muta-tions in the receptor have been found, and nearly all arein the extracellular domain and result in gross alterationin receptor structure, generally through premature ter-mination of translation. Other cytokine family recep-tors involved in disease include the leptin receptor,which is mutated in a syndrome of obesity and pituitarydysfunction, and the GCSF receptor, which is truncatedin severe congenital neutropenia. A particularly impor-tant example is mutation of the common γc chain of theIL receptor in severe combined immunodeficiency. Notsurprisingly, the downstream Jak and Stat proteins arealso likely to be targets for mutation in human disease,and Jak2 and Stat5B are both reported to act as fusionpartners in chromosomal translocations is particularforms of leukemia.

3.5. Receptors That ActivateDevelopmental Signaling Pathways

Several highly conserved signaling pathways, inaddition to those already described, play importantroles in the development of metazoan organisms andhave common features that warrant their considerationas a single group. These include DSL ligand signalingthrough the Notch receptor, Wnt signaling through theFrizzled receptor, and Hedgehog signaling throughthe Patched and Smoothened receptors. In each of thesecases, receptor activation is associated with movementof a key regulatory molecule from the cell surface orcytoplasm to the cell nucleus, regulated proteolysis isa key event in transducing the signal and genes that areotherwise repressed become activated through a core-pressor/coactivator switch.

The Notch family of receptors (Mumm and Kopan,2000) are large type I transmembrane proteins with anextracellular LBD. However, the ligands for Notch arethemselves cell-surface type I transmembrane proteinsof the Delta, Serrate, and Lag2 (DSL) family, so thatNotch mediates direct cell-cell signaling. Another sur-prising feature of the Notch receptor is that the cytoplas-mic domain, which has signaling function, appears toact in the nucleus to modulate transcription. In response

to ligand binding, proteases of the presenelin familycleave the Notch receptor, releasing the Notch intracel-lular domain. This domain has a nuclear localizationsignal, and it enters the nucleus to form a complex withproteins of the CBF-1, Su(H), Lag-1 family (CBL) andconverts these proteins from transcriptional repressorsto transcriptional activators, stimulating target gene ex-pression. Notch signaling is illustrated in Fig. 8A.

The Frizzled family of receptors (Wodarz and Nusse,1998) bind to a large array of secreted Wnt ligands tomediate diverse effects on cell proliferation and differ-entiation. The Frizzled receptors are seven-membrane-spanning domain receptors and thus can be classed withthe GPCRs, but there is still no strong evidence to indi-cate that these receptors are in fact G protein coupled.The best, although still incompletely, characterizedpathway of Wnt signaling involves the stabilization ofcytoplasmic β-catenin. In the absence of Wnts, β-cateninis phosphorylated by the kinase GSK3, which triggersits ubiquitination and degradation. This occurs within amultiprotein complex, and the activity of this complexis modulated by the Dishevelled protein, which is gene-tically downstream of Frizzled receptor signaling. In thepresence of a Wnt ligand, the activity of the complexpromoting β-catenin degradation is repressed. The neteffect is an increase in cytoplasmic β-catenin levels,allowing β-catenin to enter the nucleus and interact withthe transcription factor TCF/LEF. This interaction pro-motes the recruitment of transcriptional coregulatorsand stimulates the expression of Wnt target genes. Thispathway of Wnt signaling is illustrated in Fig. 8B. Inaddition, there are reports of Wnt signaling through path-ways as diverse as Jun N-terminal kinase activation andcalcium mobilization.

The Hedgehog family of ligands, which in verte-brates includes Sonic, Indian, and Desert Hedgehog,signals through a mechanism that is similar in manyrespects to the Wnt pathway. In this example, theHedgehog binding receptor, Patched, is a 12-transmem-brane domain protein that modulates the activity of acoreceptor, the 7-transmembrane domain Smoothenedreceptor (Ingham and McMahon, 2001). There is nostrong evidence for G protein coupling of the Smooth-ened protein, and it does not interact directly with theligand Hedgehog. Instead, Hedgehog binding toPatched appears to relieve Patched-mediated repressionof Smoothened, allowing activation of Smoothened andtransduction of the signal. Events immediately down-stream of Smoothened are unknown but act on amultiprotein complex to modify the proteolytic process-ing of a key signal transducer, a zinc-finger proteinknown as Ci (in Drosophila) or Gli (in vertebrates). Inthe absence of a Hedgehog signal, Ci/Gli is processed

ENDOCRINOLOGY · 2013-07-18 · endocrinology. The challenge in its presentation was to produce a...

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