THE HUMAN 5-HT2A RECEPTOR: GENETIC VARIATION

AND IMPLICATIONS FOR DISEASE RISK

By

Lisa A. Hazelwood

Dissertation

Submitted to the Faculty of the

Graduate School of Vanderbilt University

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in partial fulfillment of the requirements

for the degree of

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D O C TO R OF PHILO SO PHY
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in

Pharmacology

August, 2004
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Nashville, Tennessee

Approved Date:
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 UMI N um ber: 3143634

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Dedicated to God,
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my constant source of strength and inspiration.
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ii

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 ACKNOWLEDGMENTS

This dissertation work would not have been possible without the support of

so many people at Vanderbilt. First and foremost, I am indebted to my mentor,

Dr. Elaine Sanders-Bush. Her knowledge, honesty and fairness have been

critical in my training. She has been constantly encouraging no matter how bleak

the situation appeared, and has been extremely supportive of all of my scientific

pursuits. She has always been willing to help me learn and succeed, and has

encouraged me to think independently as well as critically about science. Her

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guidance and direction have certainly imparted not only a greater understanding

of and appreciation for science, but have also provided me with an example of
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how to be an excellent scientist in all respects.
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I am also extremely grateful to my committee members: my committee

chair, Dr. Randy Blakely, Dr. Rich Breyer, Dr. Ron Emeson and Dr. Richard.

Shelton. I have always walked away from my committee meetings with exciting
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new ideas to pursue and, on more than one occasion, some humorous stories as

well. They have been extremely supportive and encouraging, and have always

made themselves available for discussions outside of our scheduled committee

meetings.

The Sanders-Bush lab has been a fantastic place to do research. All of the

members of the lab, both past and present, have not only been great scientific

colleagues, but also a lot of fun to be around. From the nicknames and the

adventures of Sara Tonin to the dressing of the bat, the beef and stuff, and our

iii

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 nice little girly corner of the lab, they have all been fantastic friends and co­

workers. Scientifically (yes, we did get some science done!), 1 especially need to

thank Drs. Jon Backstrom, Lori McGrew and Paul Gresch for all of their scientific

discussions and advice, and for teaching me so many of the techniques that

were necessary for this work.

I was also able to pursue my interests in teaching while at Vanderbilt.

Many thanks go to the instructors of the Receptor Theory course for allowing me

to be the TA. Drs. Brian Wadzinski, Bih-Hwa Shieh, Seva Gurevich and,

especially Rich Breyer who originally helped me get off the ground with the idea.

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I am also indebted to Drs. Heidi Hamm and Joey Barnett for allowing me to play

a role in such an important part of the curriculum, and to Elaine Sanders-Bush for
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allowing me to spend a little less time in lab in order to gain hand-on teaching
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experience.

Last, but certainly not least, I need to thank all of my friends and family

who supported me through my seemingly endless schooling! Many thanks to all

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of my friends for providing not only encouragement but also necessary

distractions from grad school. Special thanks to Arlene, Erik & Celia, Nicki,

Elaine, Joei, Clare and Carin & Patrick for their willingness to create diversions!

My parents and brother have been my biggest advocates for so long now. Their

support has meant so much to me, and I know that I could not have

accomplished any of this without their love and support. And finally, a huge debt

of gratitude goes to my husband, Don. He has been constantly encouraging and

supportive, and has made every effort to help me through my research, even if it

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 meant hitting the bench along side me! I am especially grateful to him for all of

the traveling he has done to visit me during the past ten months while all of this

work was coming to a close. His love and support, and most significantly his faith

in my abilities, have truly lifted my spirits and encouraged me. He has helped me

to keep everything in perspective and to keep my eye on the finish line - and I’ve

finally made it!

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 TABLE OF CONTENTS

Page

DEDICATION.................................... ii

ACKNOW LEDGMENTS................................................................................... iii

TABLE OF CONTENTS................................................................................................ vi

LIST OF TABLES......................................................................................................... viii

LIST OF FIG U R E S ........................................................................................................ ix

LIST OF ABBREVIATIONS ...... xi

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Chapter

I. INTRODUCTION.........................................................................................................1
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Serotonin: Function and History.......................................................................1
Serotonin Receptor Subtypes.............................................. 2
Serotonin 5 -HT 2 Receptors.............................................................................. 5
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5-HT2a/2c Receptors........................................................................................ 10
Receptor Distribution.....................................................................19
Signal Transduction.......................................................................19
Pharmacology of 5 -HT 2A/2C Receptors........................................20
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Receptor-Ligand Interactions of 5-HT2a/2c Receptors.............. 22

Structural Domains of S -H T ^ c Receptors...............................25
Mechanisms of Receptor Regulation............................................................31
Receptor Activation of G-Proteins................................................31
Mechanisms of Receptor Desensitization.................................. 32
Recovery of Receptor Response.................................................37
5-HT2a/2c Receptor Regulation.................................................... 39
Molecular Diversity of 5-HT2a/2c Receptors..................................................42
RNA Splicing and RNA Editing of 5-HT2A/2C Receptors...........42
Single Nucleotide Polymorphisms ............ 45
5-HT2c Receptor SNPs................................................................. 47
5-HT2A Receptor S N Ps................................................................. 53
Psychiatric Disorders and the Serotonergic System.................................. 56
Major Depressive Disorder ....... 58
Specific Aim s................................................................................................... 62

II. FUNCTIONAL ANALYSIS OF NON-SYNONYMOUS SNPs: HIS452TYR

vi

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 POLYMORPHISM IN THE HUMAN S-HTaA RECEPTOR
DESTABILIZES THE SIGNALING CONFORMATION....................... 64

Introduction...................................................................................................... 64
Materials and Methods................................................................................... 65
Materials............................ 65
Site-directed Mutagenesis and Subcloning................................ 65
Cell Culture........................ 67
Radioligand Binding...................................................................... 67
Surface Biotinylation..................................................................... 69
[35S]-GTPyS Assay....................................... 70
Phosphoinositide (PI) Hydrolysis.................................................71
PLD Assay......................................................................................72
Results.............................................................................................................. 72
Wildtype and Variant Receptors are Expressed
at Similar Levels............................................................................ 72
H452Y Variant Receptors do not Couple Effectively

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to G-Proteins...................................................................................74
Variant Receptors Fail to Generate a Maximal Response
Following Agonist Stimulation...................................................... 77
Desensitization of the H452Y receptor variant is altered 85
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Constitutive activity of the 452Tyr variant is unchanged 86
Discussion........................................................................................................94
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III. GENOTYPIC ANALYSIS OF NON-SYNONYMOUS 5-HT2A RECEPTOR
SNPs IN PATIENTS WITH MAJOR DEPRESSIVE D IS O R D E R 98

Introduction.......................................................................................................98
Methods........................................ 99
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Subjects................................................................................... 99
DNA Extraction............................................................................ 102
DNA Analysis................................................................................102
Statistical Analysis....................................................................... 103
Results and Discussion...................................................... 103

IV. SUMMARY AND FUTURE DIRECTIONS........................................................111

REFERENCES......................................................................

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 LIST OF TABLES

Table Page

1. G-protein subtypes ......................... 33

2. Frequency and distribution of 5 -HT 2A/2C receptor SNPs................................... 48

3. Association studies of SNPs in the human 5 -H T 2a/2c receptors...................... 52

4. Subtype characteristics of major depressive disorder......................................59

5. Wildtype and variant receptor densities..............................................................73

6. Ligand affinities at wildtype and variant 5 -HT 2A receptors............................... 76

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7. Endophenotypes examined in MOD patients................................................... 101

8. Frequency of 5 -HT 2A receptor polymorphisms in an MDD population 106

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9. Allelic frequencies within endophenotypes.......................................................108
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10. Allelic frequencies and therapeutic response................................................... 109
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viii

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 LIST OF FIGURES

Figure Page

1. Serotonin receptor family.............................................................................3

2. Sequence alignment of the human 5-HT2a, 5-HT2b and 5-HT2C

receptors......................................................................................................... 6

3. Sequence alignment of 5-HT2a receptor species isoforms...................... 11

4. Sequence alignment of 5-HT2c receptor species isoforms...................... 15

5. 5 - H T ^ c receptor signaling pathways.................................... 21

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6. Class A GPCR conserved domains and residues.................................... 28

7. Human 5 - H T ^ c receptor post-translational modifications.....................30

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8. Heterotrimeric G-protein activation/inactivation cycle..............................34
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9. GPCR desensitization and resensitization................................................ 38

10. 5 -H T 2c receptor editing sites and coding S N P ..........................................44

11. Human 5 - H T ^ c receptor promoter maps and polymorphisms............. 50

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12. Human 5-HTja receptor S N P s.................................................................... 55

13. Cell-surface of the wildtype and H452Y variant 5-H T ^ receptors

expressed in NIH 3T3 cells......................................................................... 75

14. Competition binding with 5-HT in the presence of GppNHp....................78

15. 5-HT-Stimulated G-protein turnover measured by [35S]-GTPyS

binding.............................................. .79

16. 5-HT-Stimulated G-protein turnover is blocked by the 5-HT2A-se!ective

antagonist MDL100907................................................................................80

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 17. 5-HT-Stimulated PI hydrolysis at wildtype and variant receptors...........82

18. Agonist-induced activation of PLC at wildtype and H452Y 5 - H T 2a

receptors....................................................................................................... 83

19. 5-HT-Stimulated PI hydrolysis measured in the presence of the

antagonist MDL100907.................... 84

20. 5-HT-lnduced activation of PLD at wildtype and variant H452Y

receptors................. 87

21. Agonist-induced desensitization of wildtype and H452Y variant

receptors....................................................................................................... 88

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22. Receptor response after short- or long-term desensitization..................90

23. Competition binding and PI hydrolysis with H452F mutant receptor....92

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24. Inverse agonist treatment of wildtype and 452Tyr variant receptors....93
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25. Pyrosequencing method for genotypic analysis......................................104

26. Pyrogram and mini-sequence report for the H452Y S N P ..................... 105
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 LIST OF ABBREVIATIONS

5-HT Serotonin; 5-hydroxytryptamine

5-HT2aR Serotonin 5 -HT 2A receptor

5-HT2CR Serotonin 5-HT2c receptor

ANOVA Analysis of variance

Ca2+ Calcium

CAMKII Caicium-calmodulin kinase II

cGMP Guanosine-3’,5’-cyclic monophosphate

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CNS Central nervous system

CT Carboxyl terminus
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DAG Diacyl glycerol
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DMEM Dulbecco’s modified Eagle’s medium

DOI (±)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane
m

Concentration of drug necessary for 50% maximal response

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FBS Fetal bovine serum

GDP . Guanosine 5’-diphosphate

GEF Guanine nucleotide exchange factor

G-protein Guanine nucleotide binding protein

GPCR G-protein coupled receptor

GTP Guanosine 5’-triphosphate

HBSS Hank’s balanced salt solution

IPs Inositol 1,4,5-trisphosphate

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 LSD Lysergic acid diethylamide

MAPK Mitogen activated protein kinase

MDL1 00907 (R)-(+)-a(2,3-dimethoxyphenyl)-1-[2-(4-fIourophenyl)ethyI]-4-

piperidinemethanol

NT Amino terminus

PDZ domain PSD-95, Dig, ZO-1 domain

PI hydrolysis Phosphatidylinositol hydrolysis

PIP2 Phosphatidylinositol 4,5-bisphosphate

PKC Protein kinase C

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PLA2 Phospholipase A2

PLC Phospholipase C
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PLD Phospholipase D
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PTX Pertussis toxin

SNP Single nucleotide polymorphism

TM Transmembrane domain
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 CHAPTER I

INTRODUCTION

Serotonin: Function and History

Serotonin is a major indoleamine neurotransmitter that was first purified

from serum and identified as one of the major vasoconstricting substances in the

periphery in 1948 (Rapport, 1948). In addition, serotonin (5-hydroxytrptamine,

5-HT) was found to regulate nonvascular smooth muscle contraction, platelet

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aggregation and gastrointestinal function. Serotonin also plays an important role

within the central nervous system (CNS). The brain serotonergic system
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regulates a variety of physiological functions including appetite, sleep, pain,
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aggression and mood. Given the important role of 5-HT in mediating normal

physiologic effects and the wide distribution of 5 -H T 2a receptors within the central

nervous system, it is not surprising that the 5-HT system has been implicated in a
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variety of psychiatric and neurological disorders, from depression and

schizophrenia to migraine and alcoholism (Hoyer et al., 1994). Although a wealth

of evidence implicates fundamental roles for 5-HT in neurotransmission, we still

do not understand the specific nature of the contribution of 5-HT to biological

functions and human disease.

The importance of the serotonergic system is broad - 5-HT and its

receptors can also be found in lower organisms, including Drosophila

melanogaster, Aplysia californica (molusks) and Caenorhabditis elegans

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 (nematodes) (Weiger, 1997). The use of these lower organisms as model

systems has greatly enhanced our understanding of 5-HT functions in higher

order systems, and will undoubtedly provide further insight into 5-HT-mediated

disorders.

In mammals, the serotonergic system sends projections throughout the

central and peripheral nervous systems; 5-HT receptors can also be found within

non-neuronal systems as diverse as the gut, platelets and cardiac tissues. Within

the CNS, serotonergic cell bodies are localized to the Raphe nuclei and the

central superior nuclei (Azmitia, 1987). From these nuclei, the serotonergic

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system projects axons throughout the brain, innervating virtually every brain

region including the frontal cortex, amygdala, olfactory bulb, cerebellum,

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hippocampus, substantia nigra and thalamus.
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Serotonin Receptor Subtypes

The diverse physiological functions of 5-HT are mediated by a large family

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of 5-HT receptors, of which there are currently 14 distinct subtypes distributed

throughout the periphery and the CNS. Thirteen of these 14 subtypes belong to

class A of the superfamily of G protein coupled receptors (GPCR) and activate a

multitude of intracellular signaling events (Fig. 1); the one remaining receptor

subtype, the 5 -HT 3 receptor, is a ligand-gated ion channel. Classification of the

GPCR 5-HT receptors into subfamilies is based upon pharmacology, sequence

homology and signal transduction properties. These criteria have established six

subfamilies of 5-HT receptors, each with a distinct pharmacology, distribution and

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 5-HT

5-HT^R 5-HT2R 5 -HT 3 R 5-HT4R 5-HT5R 5-HT6R 5-HT7R

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5-HT1A 5"HT2A 5-HT5A
5-HT1b/0

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5-HT2B 5-HT5B
5-HT1d 5-HT2C

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5-HT1e
5-HT1f
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Inhibition Activation of Ligand-gated Activation Inhibition Activation Activation
of adenylyl phospholipase ion channel of adenylyl of adenylyl of adenylyl of adenylyl
cyclase C-(3 cyclase cyclase cyclase cyclase

Figure 1: Serotonin receptor family

 function.

The 5-HT-i receptor subfamily consists of five receptor subtypes and

shares 40-63% sequence identity. These receptors preferentially couple to G,y0,

negatively regulating adenylyl cyclase activity (Hoyer et al., 1994). The 5-HTi

receptor family is widely distributed, with receptors located throughout the

periphery and the CNS, including the auto-regulatory 5-HTi receptors in the

raphe nucleus.

The 5-HT4, 5-HT6 and 5-HT7 receptors all couple predominantly to Gs to

positively regulate adenylyl cyclase activity, but differences in pharmacology and

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sequence homology dictates classification in separate subfamilies (Hoyer et al.,

1994). The 5-HT4 receptor has seven splice variants, all with similar properties.
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This receptor is distributed throughout the CNS and periphery, including the gut,
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heart and vascular smooth muscle. The 5-HT6 receptor consists of only one

subtype and expression is restricted to the brain. The 5-HT7 receptor has four

splice variants. This receptor is predominantly expressed in the vascular and

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non-vascular smooth muscle; there is also limited expression of the 5-HT7

receptor in the brain.

The 5-HT5 receptor subfamily is still being functionally characterized. To

date, two subtypes of this receptor, the 5-HT5a and 5-HT5B receptors, have been

examined. Interestingly, the 5 -H T 5b receptor appears to have been lost during

evolution; the homologue of this receptor exists in the human genome, but

expression and function have not been demonstrated (Grailhe et al., 2001). The

human 5-HT5A receptor couples to both Gi/0 and Gq/n (Noda et al., 2003),

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 resulting in negative regulation of adenylyl cyclase and inwardly rectifying sodium

channels. The receptor-G protein coupling profile may be affected by expression

patterns; to date, 5-HT5a receptor expression has only been shown in the brain.

The final 5-HT receptor subfamily is the 5-HT2 receptor group. These

receptors, specifically the 5 -H T 2a and 5-HT2C receptors, will be the focus of the

remainder of this work.

Serotonin 5 -HT 2 Receptors

The 5-HT2 receptor subfamily shares 46-50% sequence identity (Fig. 2)

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and consists of the 5-HT2A, 5-HT2B and 5-HT2c receptors. In addition to sequence

homology, this receptor family is also characterized by preferential coupling to

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the G-protein Gaq and shared pharmacological properties.

The 5-HT2a receptor was first identified as the D, or dibenzyline-

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responsive, receptor by Gaddum and Picarelli (Gaddum and Picarelli, 1957).

However, this initial receptor classification was limited and, as radiolabeled

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ligands became more widely available, the D receptor was renamed the 5-HT2

receptor based upon affinity for and displacement of radiolabeled drugs

(Peroutka and Snyder, 1979). With the advent of molecular cloning and

sequencing, the 5-HT2 receptor cDNA was first correctly isolated from rat brain

(Julius et al., 1990) and later cloned from humans (Saltzman et al., 1991). The 5-

HT2b receptor was first identified in the stomach fundus and was cloned and

classified as the 5-HT2F receptor (Kursar et al., 1992). The 5 -H T 2c receptor was

characterized in rat choroid plexus, and was named the 5-HT-ic receptor, as a

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 Consensus MXXLXXXVXSLXSTXXXLXXXXXDXXVXS-

5 -H T 2AR L|C E E N T ; a..L[S|.S f f l T N S jjjM Q L N q jjp F R L T Y ] S

5-HT2BR L S Y R S T I P E H I L Q S T F V H
5-HT2CR xJ r n a I LVWQCfilS

Consensus ------------- X X A N X S D X F N X T X D X E X R X X X X X X G -

5"HT2AR D F N S G E I A NpTj S I^ F ^ J f W [T [ ^ J J S l E fW jR jT N L S C cl
5"HT2BR ------------------- USsN wj SfG L Q“ T E S I P E E M K Q I V E E
5"HT2CR ------------- V A|A [ l V q D| I | -[T | s [6 ) g - GjR|F K F P D G

Consensus

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X X -------------------------------------- X N W X A L L I X X V I I X T I G

5 -H T 2AR L S P S C L S L L H L Q E K N W T tg
5-HT2BR N K ---------------------------------------L Hj:li ALL! ¥11 T I G
5"HT2CR
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V Q ----------------------------------------- rN W V I I J I TIG'
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Consensus G N I L V I M A V S L E K K L Q N A T N Y F L M S L A I A D

5-HT2AR G N I L V 1 M A V S L E K K. h Q N A T N Y F L M S L A I A D:
5"HT2BR G N; T| L V I j L: A V S T, E K K L Q\ Y| A T N Y F 1, M S L A[V| A D
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5 -H T 2CR G N 1 L V I M A V S' S E K K L g N A T N Y F L M S L A I A D

Consensus M L V G L L V M P X S L L T I L Y X Y X W P L P X X L C P V

5 -H T 2AR M L jL j G[F; I j V M PfVi S[M) L T I L ^ G l Y|R| W P L P f S K| L CjA| V

5-H T 2BR l LI L V G L)'Fj V M pj I A 1, L T 1| M F E A Ml W P L P L v j L C p| Aj
5 -H T 2CR ¥ I j V G L E V M Pj L jS L L] A j ' T f | D ; Yj vj W P L P|R Y: L C P V ;

Figure 2: Sequence alignment of the human 5-HT2A, 5-HT2B and 5-HT2C receptors.

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 Consensus W I X L D V L F S T A S I M H L C A I S L D R Y V A I X N P

5-HT2AR W JjY l L D V L F R T A M T, C A '■ o L D P Y V A I Q|N P

5-HT2BR Wj L F j l D V L F S T A M i! L C A I V: D R Y j E A T K Kl P
K I SiLDVLF S T A S I M H h C A I *
o L D R y v a 1 Rl n p
5-HT2CR

Consensus i x h s r f n s r t k a f x k i x x v w x i s i g i s x p i

5-HT2AR H H 3 R f n i R T K A F "L K A; V WI T I Si'ViG I SjM p i

I iI
Q A N Q Y IN •: R |A Tj A F I K I j T V: V Wj L I S I G I [A I PjVl
5 -H T 2BR
E H S R F N G U I ’ K A I' M K I | V W{A T S I GjVj S ]V p lT
5-HT2CR }\L

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Consensus p v x g l x d d x k v f x n - x t c v l x d x n f v l

5 -H T 2AR Q] D DfSj K V F [ K f G S | C [ g L [ A j^D [ D F V J.

5-HT2BR V D N P N ® T C V L : T ' K E R F G D S p[M] Ij

5 -H T 2CR
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E e Hr V F | V N N T T C V . L Np ] p p V L
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Consensus i g s f v a f f i p l t i m v i t y f l t i x x l q k x a x

5-HT2AR 1 G S F Vi S : F F I P L T 1 M V I T Y F 1. T K S I j Q K 'E 7r
5 -H T ,r R j'FjG S I. A rA F F [T j P L|A ] I Mi I Vi T Y F L T H A L Q K K A Y
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i G !'■ F V A F F 1 P L T 1 M V i ' t Y | C | L T Y V L IR R Q A L
5-HT2CR

Consensus l x x x x x x x r x x x l s x s f l x x x x x x x x x ---------

A K L Am P -------------------------------------
5 -H T 2AR
5 -H T 2BR V K N K B T V F Q R D E T P C S S P E
HGH Si L D K C C K R N T A E ---------
5-HT2CR

Figure 2, cont.

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 Consensus X X X X A X P N X X Q X X X X R X X X S X X

5-HT2AR Q S^STTS" S E K f T F ] 0 | R S I Ef R E P G Y T
5 -H T 2BR K V A M L D G S R K D K P N S G D E T L M IR R T I
5-HT2CR ------------------------------ E E N S P N Q D Q N A R R| R K K K E R R

Consensus g r x t m q x i s n e q k a s k v l g i v f f l f l x m w c

5-HT2AR G R jR ; T M Q S \ l S N K Q K A | C j K V L O I V F P L F f V Vi M W C!
5-HT2BR G [ K K S' V y T I S N E QjRj A S K V L G- I V F F L F LG., M W C
5-HT2CR [p ]R |g ;T m y I [N; N EfRj'K A S R V L G I V F F[Vj F I,] l \ M W

Consensus

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p f f i t n i x x v l c x x s c n q x x x x x l l n v f v w

5 -H T 2AR I T N M A V fjg c jT T E s c n \ E _ D V I G A h h v f v w
5-HT2BR X T N T L V I C D - s SI 3S - T T L Q M L L E ~ ll F V w

5 -H T 2CR
I T*N
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L S V L c \ E K s c n Q K L M E K L' L, N V F V' W
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Consensus i g y v s s g v n P L V Y T L F N S R Y I X C

5-HT2AR P L V Y T L }' N K 7 F S] A F S a Y: X Q
5-HT2BR P L V Y T L 1 N K. T f F : R D| A ‘F[G] R Y 1 T
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I G Y V[ C S G j I~! N P L V Y T L F N K fl'i'Y R R A F S j N j Y L R
5-HT,rR
r2cf

Consensus n y k x x k - K P X X X X I X R N X X X A L S X X

5-HT2AR [Q'jY KjE~N K - L Q L| tJTTV]S} T I P A L A Y K

5-HT2BR N Y j~RJA T K S V K T L R r s s k i T f|r k P M A E N S K F
N Y ’K iV E K - - - - - - k A A T|AL $ G R
5-HT2CR A

Figure 2, cont.

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 Consensus X X X X X I X X X X N -------- S P X X X K X S D N X X S X

5-HT2AR S S Q L Q M G Q K K n F 'ill K Q D A
5-HT2BR F K K H G 1 R N G I P A M Y ]MRL
E L N V N Y R H T _EpKjA, 53 D N | E P G
5-HT2CR

Consensus x x l — x x x l e l x x n x s d x v x e r v s x v

5-HT2AR v ~ a ] l J g k q h s e: E | a s k T d ”n] S D|~G|VfNl B [ K ] V S|

5 -H T 2bR I l | L j - - l d t LjJLJL Dj K T E; E R ? ' S
E M Q V E N £i E Ic S V py jS iE R [T jS
5 -H T 2CR

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EV
PR

Figure 2, cont.

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 member of the 5-HTi receptor subfamily (Pazos et al., 1984).

The rat 5-HTic receptor was first cloned in 1988 (Julius et al., 1988), and

cloning of the human isoform soon followed (Saltzman et al., 1991). Molecular

cloning allowed direct sequence comparison of the 5 -HT 2, 5-HT2f and 5~HTic

receptors, revealing high sequence homology. This sequence similarity,

combined with similarities in pharmacology and signal transduction properties,

prompted the existing subfamilies to be reclassified into their current group: the

5-HT2 receptor was renamed 5 -H T 2A, the 5-HT2F renamed as 5-HT2B, and the 5-

HT-tc became the 5 -H T 2c.

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5-HT2A/2C Receptors
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The htr2a gene encoding the 5-HT2A receptor has been mapped to human

chromosome 13q14-q21; the coding region of the gene is separated by two

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introns and spans 1.4 Kb encoding 471 amino acids. The rat, mouse, monkey

and pig homologues are also each 471 amino acids and share a great deal of
PR

sequence similarity with the human receptor subtype (Fig. 3).

The 5 -H T 2c receptor, encoded by the htr2c gene, contains three introns

and has been mapped to human chromosomal location Xq24. The coding region

of the 5 -H T 2c receptor spans approximately 1.4 Kb and encodes a protein of 460

amino acids. 5-HT2c receptors share a high level of sequence identity across

species (Fig. 4), although there is slight species variation in the length of the

5 -H T 2c receptor protein. The 5-HT2c receptor is also quite homologous to the

5-HT2a receptor, with the two proteins sharing greater than 50% overall

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
 10 20 30

H um an V D 1 . c E E N T »S S r> r : N S M Q 1., N :: D T
; R i £3 N
Monkey M d T L C E E N T' s (fi y T t N ;S f' V i, <■! E D T R L £3 N
P ig M d v ;i. r E J-J N 1" : i, ;■\c <%
> a ip 'i N ) F M Q L ;i D C T R i. Hj N
Mouse M E T «■ F. D N I a ■;jy; a i!Si ii i pi it a a I.. G n 11 S & L Y P jN
ij.lr .;;
Rat M E I :3! r F; D N I s 'ii£? ! T p N s iiii M L- G D G. P B i if! H j N

40 50 60

Human D F h a G E A N T : D A ' r-. w T V n 55 F. N P T N I. i ‘ F. G r

Monkey D F N ItH G K A N T 5' 1.) .a E y E N 1? T
N w T V M 1. li ij 3i i-
Pig i: F N H {3 E (Si N T pi !fflf A K M M i l?i 1) s k >' R T H 1 F E ,

Mouse I; F N i t 1 R D - N ■J f; E A s N ¥! T r D A E N R T I-! L S C E G | Y -
Rat I) F N R D A N T 53 E W T i :1S A | K IJ R T I: h ipiii E G[Yj
A S f-

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70 80 90

Human 1, S p S c L S J, i„ H K K N 3 A 11 L T V '■ 1 r i, T 1
Monkey L p s "iffl L s I. Ij " L Q E K N Y c A ]., h T A SI v I r i, T 1 A
Pig t. s p s F I £5 r1.
IE L H I, E K N jp i ■\ i .j I, T i - V :r i h T 1 A
Mouse L ■pi p T G i i £3 Ij It ||| Q E K N W S A L h M’ I'T j M V t i I.. ! A
Rat h I> I 3 2 r I j £5 ■ I . f f i H L Q E K N S:i L L T !T j V V i i. i, 1 A
L ‘M i
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100 110 120

H u m a n G N 1 L v ' 1. M A " \ T s L E K K I, Q N A. T W Y F I , I'd" y 1, A I A D ,
M o n k e y G N I h V .r M ft. V 53 L . E K K L Q N A T N Y F L M a I. A 1 A D
Pig G N I 3_- V j" M A V 33j, E k Y h Q N A T N Y F 33 M S 3, 'A I. A P
PR

M ouse gw i h v j m a v a l e k k l q n a n y f l m a l a . i a d
Rat GN 3 li V I M A V ;3 I. E K k L g W A T . 1-3 Y ¥ L M G E A J A D

130 140 150

Human M L I, ’ G F L V MF V S ' M L T T I., Y ~G Y R W P I 1; E K L C A V
M onkey m L L '* F v w p v s M l T i: l y g y r w p l p s k l <: a v
Pig M L 3j G F 1', V M P V S M 3, I1 J L Y G Y R W P I, P 3 K I, C A V
M ouse M 1- E G F 1, V 14 P '•/ ;.I M L T I 3. Y G Y k W P L P H K 3, C’ A V
R at M L E G F 1, V M P V ' M L T J. I. Y G Y R W P L P ,0 K L A flj

Figure 3: Sequence alignm ent o f 5-HT2A receptor species isoforms.

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