Lennarz W.J., Lane M.D. (eds.) Encyclopedia of Biological Chemistry. Four-Volume Set . V. 4

Подождите немного. Документ загружается.

suppressing transcriptional activation through the

NFAT:AP-1 complex.

Similar dual pathways involving PP2B and NFAT

family members have been implicated in angiotensin-II-

induced cardiac hypertrophy and in the morphogenesis

of heart valves.

DARPP-32

DARPP-32 (dopamine and cyclic adenosine 3

-mono-

phosphate-regulated phosphoprotein, 32 kDa) is a speci-

ﬁc inhibitor of PP1. It is expressed at high concentrations

in medium spiny neurons of the neostriatum where it

plays a central role in integrating responses to dopamine

(acting via the cAMP) and glutamate (acting via Ca

2þ

)

in dopaminoceptive neurons (Figure 3, right). Diseases

associated with defects in dopaminergic neurotrans-

mission include Parkinson’s disease, Huntington’s dis-

ease, ADHD, and schizophrenia.

Activation of the cAMP pathway in dopaminoceptive

neurons leads to increased phosphorylation of

DARPP-32 on Thr 34 and inhibition of PP1. This results

in increased phosphorylation of neurotransmitter recep-

tors, voltage-gated ion channels, an electrogenic ion

pump (Na

ATPase), and a transcription factor

(CREB).

In contrast, glutamate acting through NMDA

receptors promotes dephosphorylation of brain pro-

teins through activation of a protein phosphatase

cascade involving PP2B and PP1. Activation of

NMDA receptors elevates Ca

2þ

which activates

PP2B. This leads to DARPP-32 dephosphorylation by

PP2B and activation of PP1 through relief of inhibition

by DARPP-32.

FURTHER READING

Barford, D. (1996). Molecular mechanisms of the protein serine/

threonine phosphatases. TIBS 21, 407–412.

Ceulemans, H., Stalmans, W., and Bollen, M. (2002). Regulatory-

driven functional diversiﬁcation of protein phosphatase-1 in

eukaryotic evolution. BioEssays 24, 371–381.

Cohen, P. (2002). Protein phosphatase 1 – targeted in many directions.

J. Cell Sci. 115, 241–256.

Greengard, P., Allen, P., and Nairn, A. (1999). Beyond the dopamine

receptor: The DARPP-32/protein phosphatase-1 cascade. Neuron

23, 435–447.

Ingebritsen, T., and Cohen, P. (1983). Protein phosphatases: Properties

and role in cellular regulation. Science 221, 331–338.

Janssens, V., and Goris, J. (2001). Protein phosphatase 2A: A highly

regulated family of serine/threonine phosphatases implicated in

cell growth and signalling. Biochem. J. 353, 417–439.

Rao, A., Luo, C., and Hogan, P. (1997). Transcription factors of the

NFAT family: Regulation and function. Annu. Rev. Immunol. 15,

707–747.

Rodriquez, P. (1998). Protein phosphatase 2C (PP2C) function in

higher plants. Plant Mol. Biol. 38, 919–927.

BIOGRAPHY

Thomas S. Ingebritsen is an Associate Professor in the Department of

Genetics, Development and Cell Biology at Iowa State University. His

research interest is the structure, regulation, and function of protein

phosphatases. He holds a Ph.D. in Biochemistry from Indiana

University and received his postdoctoral training at the University of

Dundee, Scotland. Together with Philip Cohen, he developed the

scheme for classiﬁcation of protein serine/threonine phosphatases and

he has published extensively in the area of protein phosphorylation and

protein phosphatases.

32 SERINE/THREONINE PHOSPHATASES

Serotonin Receptor Signaling

Paul J. Gresch and Elaine Sanders-Bush

Vanderbilt University, Nashville, Tennessee, USA

Serotonin (5-hydroxytryptamine; 5-HT) receptors are a family

of G-protein-coupled receptors (GPCRs) and one ligand-gated

ion channel that transduce an extracellular signal by the

neurotransmitter serotonin to an intracellular response. 5-HT

receptors are involved in multiple physiological functions such

as cognition, sleep, mood, eating, sexual behavior, neuroendo-

crine function, and gastrointestinal (GI) motility. Since many

physiological processes are inﬂuenced by 5-HT receptors, it is

not surprising that dysfunction and regulation of 5-HT

receptors are implicated in numerous disorders and disease

states including migraine, depression, anxiety, schizophrenia,

obesity, and irritable bowel syndrome. Therefore, under-

standing 5-HT receptor second messenger systems, their

effector linkage, the multiplicity of coupling pathways, and

how these pathways are regulated is critical to disease etiology

and therapeutic discovery.

Serotonin Synthesis

and Metabolism

The neurotransmitter serotonin (5-hydroxytryptamine;

5-HT) is found in the central nervous system, entero-

chromafﬁn cells, gastrointestinal tract, and platelets.

5-HT is synthesized from the essential amino acid

tryptophan by the rate-limiting enzyme tryptophan

hydroxylase and a ubiquitous l-aromatic amino acid

decarboxylase. 5-HT is released into the synaptic cleft by

exocytosis of vesicles in a TTX-sensitive and Ca

2þ

dependent manner. Inactivation of 5-HT is mediated by

reuptake into the presynaptic terminal through an Na

dependent 5-HT transporter. 5-HT is metabolized into

the inactive form, 5-hydroxyindole acetic acid, by the

enzymes monoamine oxidase and aldehyde dehydro-

genase. Levels of synaptic 5-HT can be regulated. For

instance, restriction of dietary tryptophan or chemical

inhibitors of tryptophan hydroxylase reduce brain levels

of 5-HT, while selective 5-HT reuptake inhibitors such

as ﬂuoxetine (Prozac) increase the amount of synaptic

5-HT. Released serotonin acts on multiple 5-HT

receptors found throughout the body in various tissues

including brain, spinal cord, heart, blood, and gut.

Serotonin Receptor Structure

and Function

5-HT receptors are classiﬁed and characterized by their

gene organization, amino acid sequences, pharmaco-

logical properties, and second messenger coupling

pathways. With the exception of the 5-HT

receptor,

the 5-HT receptor family consists of G- protein-

coupled receptors (GPCRs). The basic protein struc-

ture is predicted to contain seven transmembrane

regions, three intracellular loops, and three extracellu-

lar loops, with the amino terminus being extracellular

and carboxy terminus, intracellular (Figure 1A). These

receptors are linked to their signal transduction path-

ways through guanine nucleotide triphosphate (GTP)-

binding proteins (G protein). The sequence of events

involves the activation of the cell surface receptor by

5-HT or drugs, binding of receptor and G protein,

which promotes the exchange of bound GDP for GTP

on the G protein. The G protein is comprised of

-subunits; the

dimer dissociates when receptor

binds and both G

and G

have the ability to

interact with effector enzymes. The subunit inter-

actions promote activation or inhibition of adenylate

cyclase or activation of phospholipase C. In turn, the

effector enzymes generate second messengers that

regulate cellular processes such as Ca

2þ

release, and

protein kinases and phosphatases. This multistep

enzymatic process ampliﬁes receptor signal, and

provides the possibility of regulation and crosstalk at

multiple levels. The numerous 5-HT receptors are

grouped in Table 1 by their traditional (primary) G

protein second messenger linkage. The multiple levels

of diversity generated by RNA splicing, RNA editing,

and promiscuity of receptor G-protein activation are

discussed later.

Serotonin Receptors that Inhibit

Adenylyl Cyclase

The ﬁve members of the 5-HT

receptor family (5-HT

5-HT

, 5-HT

, and 5-HT

) and the 5-HT

receptor (5-HT

, 5-HT

subtype) couple primarily

through G

i/o

proteins to inhibit the membrane-bound

enzyme, adenylyl cyclase (AC). This inhibition of AC

leads to a decrease of 3

-adenosine monophosphate

(cAMP) molecules (Figure 1B). The 5-HT

receptors are

the best characterized of this family. High densities of

5-HT

receptors are found on the cell bodies of 5-HT

neurons in the brainstem nuclei, especially the dorsal

raphe. In the dorsal raphe, the 5-HT

receptor

functions as an autoreceptor that reduces cell ﬁring

when activated. The receptor elicits neuronal membrane

hyperpolarization by activating G protein-linked K

channels. In addition, 5-HT

receptors are located on

postsynaptic sites in the hippocampus and other limbic

brain regions where they also produce hyperpolarization

by opening K

channels. 5-HT

receptors are targets of

a class of antianxiety drugs. Of the other 5-HT

receptors, much more is known about the 5-HT

and

5-HT

subtypes. These receptors are found in basal

ganglia and frontal cortex, and function as terminal

autoreceptors or heteroreceptors that modulate neuro-

transmitter release. 5-HT

1B/1D

heteroreceptors have

been proposed to regulate the release of acetylcholine,

glutamate, dopamine, norepinephrine, and

-aminobu-

tyric acid (GABA). Pharmacological and genomic

(knock out/deletions) studies suggest that 5-HT

receptors are involved in aggressive behavior. 5-HT

receptors have a role in migraine headaches and many

antimigraine drugs target this receptor. Less is known

about 5-HT

, 5-HT

, and 5-HT

receptors.

Serotonin Receptors Linked to

Activation of Adenylate Cyclase

5-HT

, 5-HT

, and 5-HT

receptors are all coupled to

activation of AC. These receptors are linked via the G

protein G

to AC producing an increase cAMP

production (Figure 1B). Historically, the ﬁrst signal-

transduction pathway to be linked to a 5-HT receptor

was stimulation of AC, characterized in mouse collicular

neurons. This receptor now known as the 5-HT

receptor is also found in hippocampus and peripheral

tissues. In the periphery, it releases acetylcholine in the

ileum, contracts the esophagus and colon, promotes ion

transport in the gut, and elicits cardiac contraction. In

the brain, the receptor has been linked to modulation of

release of acetylcholine, dopamine, 5-HT, and GABA.

Little is known about the 5-HT

receptor. It is found in

the striatum, amygdala, nucleus accumbens, hippo-

campus, and cortex. 5-HT

receptors are widely

expressed in the brain, with highest expression levels

in the thalamus and the hippocampus. The 5-HT

receptor may have role in circadian rhythms and

thermoregulation. Both the 5-HT

and 5-HT

receptor

have high afﬁnity for many of the atypical antipsychotics

leading to speculation of a role for this receptor in

schizophrenia.

TABLE 1

The Primary Signal Transduction Pathway for the Serotonin

Receptor Family

G protein

5-HT receptor

subtype

Effector

linkage

1A Inhibition of adenylate cyclase

4 Activation of adenylate cyclase

2A Activation of phospholipase C

No G protein 3 Ligand-gated ion channel

ATP

cAMP

ai/o

PLC

Heterotrimeric

G-protein

Carboxy terminus

Amino terminus

PIP

+ DAG

PKA

–

FIGURE 1 A. Schematic drawing of the basic components of a

G-protein coupled receptor illustrating the extracellular amino

terminus, seven transmembrane domains, three intracellular loops,

three extracellular loops, intracellular carboxy terminus, and hetero-

trimeric G protein. B. The primary signaling pathways of the G-protein

coupled receptors. Activation of G

protein inhibit AC resulting in the

decrease of cAMP production. Receptors that stimulate AC through

results in increased production of cAMP, with subsequent

activation of PKA. Stimulation of PLC

through G

results in the

cleavage of PIP

into IP

and DAG.

34 SEROTONIN RECEPTOR SIGNALING

Serotonin Receptors Coupled to the

Activation of Phospholipase C

The 5-HT

class of receptor (subtype 2A, 2B, 2C)

activates the membrane-bound enzyme phospholipase C

(PLC) which catalyzes the degradation of the inositol

lipid, phosphotidylinositol 4,5 bisphosphate (PIP

) with

the production of inositol 1,4,5 triphosphate (IP

)and

diacylglycerol (DAG) (Figure 1B). IP

mobilizes Ca

2þ

from intracellular storage sites; Ca

2þ

then induces

multiple responses in the cell including activation of

calcium/calmodulin-dependent protein kinase enzymes

that phosphorylate protein substrates in the cell. DAG

activates another kinase, protein kinase C. The 5-HT

receptors are coupled via the G protein G

or G

activation of PLC.

The 5-HT

receptor is involved in smooth muscle

contraction and platelet aggregation. In the brain,

5-HT

receptors are found in cerebral cortex, claus-

trum, and basal ganglia. It is thought that hallucino-

genic drugs exert their psychotrophic action by activating

5-HT

receptors. The 5-HT

receptor was ﬁrst

described from the stomach fundus and later was

identiﬁed in the gut, heart, kidney, and lung. Its presence

in the brain is less certain. The 5-HT

receptor is found

in choroid plexus, where it regulates cerebral spinal

ﬂuid production and ion exchange between the cerebral

spinal ﬂuid and brain. The 5-HT

receptor is also

found in various brain regions such as frontal cortex

and amygdala. Activation of brain 5-HT

receptors can

lead to hypoactivity and hypophagia. Moreover, atypical

antipsychotic drugs block the activation of 5-HT

and

5-HT

receptors, indicating that these receptors may

be involved in the pathophysiology of schizophrenia.

The Serotonin-3 Receptor

is a Ligand-Gated Ion Channel

The 5-HT

receptor is different from the other 5-HT

receptors in that it forms an ion channel that regulates

the ﬂux of ions. The structure of receptor is a pentamer,

similar to the nicotinic acetylcholine receptor. The

receptors are found on neurons in the hippocampus,

nucleus tractus solitarius, and area postrema, as well as

in the periphery. They are located on pre and

postganglionic autonomic neurons and alter GI tract

motility and intestinal secretion. When activated by

5-HT, the receptor triggers rapid depolarization due to

an inward current by opening a nonselective cation

channel (Na

,Ca

2þ

inﬂux and K

efﬂux). The receptor

is possibly involved in nausea, vomiting, and irritable

bowel syndrome.

Alternative Splice Variants

of Serotonin Receptors

Functional diversity of proteins can be produced by

alternative splicing events. Many of the 5-HT receptor

genes contain introns that are subject to alternative

splicing with the generation of multiple-receptor

mRNAs encoding slightly different proteins, referred to

as isoforms. Seven carboxy-terminal splice variants of

the 5-HT

receptor have been described. The most

interesting feature of these splice variants is the level of

constitutive activity of the receptor, which is markedly

increased. Constitutive activity is the ability of a

receptor to activate second-messenger pathways spon-

taneously without the binding of an external ligand.

Four carboxy-terminal splice variants of the 5-HT

receptor have been identiﬁed. The functional conse-

quence of these variants is uncertain. An alternatively

spliced variant of the 5-HT

receptor has been

described, which encodes a truncated, nonfunctional

protein. More work needs to be done to determine the

physiological relevance of these RNA splicing events;

nonetheless, is it clear that this process leads to

additional diversity in 5-HT receptor signaling.

RNA Editing Produces

Multiple Functional 5-HT

Receptor Isoforms

The 5-HT

receptor undergoes a unique process termed

RNA editing to yield multiple-receptor variants. RNA

editing is an enzymatic reaction that alters nucleotide

sequences of RNA transcripts. For the 5-HT

receptor,

ﬁve encoded adenosine residues are converted to

inosines by double-stranded RNA adenosine deami-

nases. In the human 5-HT

receptor, the adenosines

within the predicted intracellular second loop can be

converted to inosine at the RNA level resulting in

multiple mRNA species with the potential to encode

24 different protein variants (Figure 2). The extensively

edited isoforms have different abundances in brain

tissue, and the translated proteins exhibit different

binding properties, and differential activation of

second-messenger systems. For example, 5-HT exhibits

a decreased potency when activating the fully edited,

VGV, isoform of the human receptor compared with the

unedited, INI, form and there is a rightward shift in the

dose-response curve for phosphoinositide hydrolysis. In

addition, editing can alter the ability of 5-HT

receptors to couple to multiple G-proteins. For example,

the non-edited 5-HT

receptor functionally couples to

and G

, whereas the edited 5-HT

receptors has

less coupling to G

. RNA editing may have clinical

SEROTONIN RECEPTOR SIGNALING 35

signiﬁcance; recent studies have indicated that altera-

tions in the editing proﬁle of the 5-HT

receptor are

associated with the incidence of suicide and

schizophrenia.

Single Nucleotide Polymorphisms

Occur in the Serotonin

Receptor Family

Normal genetic variations (single-nucleotide poly-

morphism, SNP) have been identiﬁed in almost all

5-HT receptors. Polymorphisms in the coding region

of the gene have the potential to alter the receptor’s

ability to bind ligand, to activate signal-transduction

pathways, or to adapt to environmental inﬂuences.

For example, a polymorphism in the amino terminus

of the human 5-HT

receptor attenuates the down-

regulation and desensitization produced by the agonist

8-OH-DPAT. A polymorphic variant in the 5-HT

receptor in the putative third transmembrane domain

alters the binding of the antimigraine drug, suma-

triptan. In addition, a polymorphism in the carboxy

terminus of 5-HT

receptor reduces the receptor’s

ability to mobilize internal Ca

2þ

. Currently, efforts

are being made to link the occurrence of 5-HT

receptor polymorphisms with various pathological

disorders. Future progress in pharmacogenomics

(using genetic information to predict drug response)

may potentially lead to better design of serotonergic

drugs to reduce side effects and target subpopulations

of patients with speciﬁc therapies dependent on their

genetic proﬁle.

Promiscuous Coupling and

Crosstalk between 5-HT Receptor

Signal-Transduction Pathways

Promiscuous coupling is the ability of a receptor to

couple to more than one signal transduction pathway.

For example, the 5-HT

receptor can both inhibit and

activate AC. As mentioned above, the primary coupling

of this receptor is G

i/o

with subsequent inhibition of

AC; this has been demonstrated both in vivo and in

cultured cell systems. However, this receptor has been

shown to activate AC, mediated by

subunits released

from G

i/o

, rather than G

protein. This activation

seems to require high receptor occupancy and high

expression levels in cell expression systems. In addition,

depending on cell type and experimental conditions, the

5-HT

receptor can activate or inhibit PLC. Many

studies suggest that the G-protein

-subunits play a

role in such crosstalk between signaling pathways. The

5-HT

receptor (as well as the 5-HT

receptor) is

another promiscuous receptor that may activate mul-

tiple signal transduction pathways. The primary coup-

ling of 5-HT

receptors is to activate PLC activity via

proteins to produce IP

and DAG. However, this

receptor can activate other phospholipases, for example,

phospholipase D through G

proteins and free

-subunits. Phospholipase D can inﬂuence many

neuronal functions including endocytosis, exocytosis,

vesicle trafﬁcking, and cytoskeletal dynamics. In

addition, 5-HT

and 5-HT

receptors can activate

phospholipase A-2 activity, which leads to the release

of arachidonic acid and potential formation of bio-

active eicosanoids such as the prostaglandins. There is

evidence that 5-HT

and 5-HT

receptors differen-

tially activate PLC compared to PLA2, dependent on

the speciﬁc agonists employed. Moreover, there may be

differences in the desensitization of these two pathways

after repeated agonist treatment. The release of arachi-

donic acid can result in the activation of K

channels,

regulation of neurotransmitter release, and can be a

retrograde messenger. It is becoming apparent that

speciﬁc 5-HT receptors interact with many G proteins,

thereby regulating multiple signal-transduction path-

ways. This diversity may be a target for future

therapeutics and interventions.

Summary: Potential Role

of Receptor Diversity

There has been much speculation on the origin and

signiﬁcance of the 5-HT receptor diversity. One possible

explanation for the numerous subtypes and variants is

that the 5-HT neurotransmitter system emerged early

FIGURE 2 The positions of the editing sites within human 5-HT

receptors RNA and amino acid sequences are shown for hINI, hVSV,

and hVGV edited isoforms of the 5-HT

receptor. These editing sites

are located in the putative second intracellular loop.

36 SEROTONIN RECEPTOR SIGNALING

during evolution. Thus, there has been ample time for

genetic variation and divergence to occur in the genetic

encoding for the receptor proteins. Whatever the process

of receptor diversity, the multiple receptor signaling has

considerable biological signiﬁcance. For example, inputs

to the dorsal raphe nucleus are integrated into global

5-HT release, via both synaptic and volume trans-

mission, that in turn can modulate multiple and diverse

neuronal functions based on receptor subtype. Thus, one

transmitter (5-HT) with multiple receptors/multiple

pathways can introduce levels of complexity, yet speciﬁc

physiological responses, that are localized to a given

brain region or neural system. These responses can be

cell-type speciﬁc or even cell-compartment speciﬁc.

Furthermore, a level of ﬁdelity is introduced based on

the receptor’s relative afﬁnity for 5-HT. The exact nature

of this diversity is at present unclear however the

physiological implications are quite apparent, when

considering the diverse role of these receptors in many

physiological functions and disease states.

FURTHER READING

Aghajanian, G. K., and Sanders-Bush, E. (2002). Serotonin. In

Psychopharmacology: The Fifth Generation of Progress (K. L.

Davis, D. Charney, J. T. Coyle and C. Nemeroff, eds.) Lippincott,

Williams, and Wilkins, Philadelphia.

Barnes, N. M., and Sharp, T. (1999). A review of central 5-HT

receptors and their function. Neuropharmacology 38, 1083– 1152.

Hoyer, D., Clarke, D. E., Fozard, J. R., Hartig, P. R., Martin, G. R.,

Mylecharane, E. J., Saxena, P. R., and Humphrey, P. P. A. (1994). VII.

International Union of Pharmacology classiﬁcation of receptors for

5-hydroxytrytamine (serotonin). Pharmacol. Rev. 46, 157–203.

Meltzer, H. Y. (1999). The role of serotonin in antipsychotic drug

action. Neuropsychopharmacology 21, 106S–115S.

Raymond, J. R., Mukhin, Y. V., Gelasco, A., Turner, J., Collinsworth,

G., Gettys, T. W., Grewal, J. S., and Garnovskaya, M. N. (2001).

Multiplicity of mechanisms of serotonin receptor signal transduc-

tion. Pharmacol. Ther. 92, 179 –212.

BIOGRAPHY

Elaine Sanders-Bush is a Professor of Pharmacology and Psychiatry

at Vanderbilt University School of Medicine, Nashville, Tennessee.

She earned a Ph.D. in pharmacology at Vanderbilt in 1967. Her

research focuses on serotonin receptors, applying a multidisciplinary

approach to deﬁne the role of signal transduction molecules and

posttranscriptional and posttranslational modiﬁcations that alter

receptor function. Dr. Sanders-Bush received a Merit Award from

the National Institute of Mental Health in recognition of her research

accomplishments.

Paul Gresch is a Postdoctoral Fellow in Dr. Sanders-Bush’s laboratory.

He earned a Ph.D. in cellular and clinical neurobiology from Wayne

State University in Detroit, Michigan in 1999.

SEROTONIN RECEPTOR SIGNALING 37

Siglecs

Ajit Varki

University of California, San Diego, Calfornia, USA

Sialic acid recognizing Ig-superfamily Lectins (Siglecs) are a

major subset of the “I-type lectins.” The latter are deﬁned as

animal proteins other than antibodies that can mediate

carbohydrate (glycan) recognition via immunoglobulin(Ig)-

like domains. Siglecs share characteristic amino-terminal

structural features that are involved in their sialic acid-

binding properties, and can be broadly divided into two

groups: an evolutionarily conserved subgroup (Siglecs-1, -2,

and -4) and a CD33/Siglec 3-related subgroup (Siglecs -3 and

-5 to -11). While the precise functions of Siglecs are

unknown, they seem to send inhibitory signals to the cells

that express them, in response to recognition events on

cell surfaces.

Historical Background

and Deﬁnition

Sialic acids (Sias) are a family of nine-carbon acidic

sugars that typically occupy a terminal position on

glycan chains attached to the cell surface of “higher”

animals. The immunoglobulin superfamily (IgSf) is an

evolutionarily ancient group of proteins whose appear-

ance predated the emergence of the immunoglobulins

themselves. Until the 1990s, it was assumed that IgSf

members (other than some antibodies) did not mediate

carbohydrate recognition. Independent work on CD22

(eventually Siglec-2, a protein on mature resting B cells)

and on sialoadhesin (Sn, eventually Siglec-1, a protein

on certain macrophage subsets) revealed that their ﬁrst

Ig V-set-like domains could mediate Sia recognition.

Homologous features of this V-set Ig-like domain and

the adjacent C2-set domain then led to the discovery

that two other previously cloned molecules—CD33

(eventually Siglec-3) and Myelin-associated Glyco-

protein (MAG, eventually Siglec-4)—also had Sia-

binding properties. Following consultation among all

researchers working on these proteins, the common

name “Siglec” and a numbering system were agreed

upon. Criteria for inclusion of IgSf-related proteins as

Siglecs are: (1) the ability to recognize sialylated glycans;

and (2) signiﬁcant sequence similarity within the

N-terminal V-set and adjoining C2-set domains. Evalu-

ation of the human and mouse genomes eventually

deﬁned 11 human and 8 mouse molecules that fulﬁll

these criteria. Since humans have more Siglecs than mice

and cloning of the mouse molecules initially lagged

behind, the primary numbering system is based on the

human molecules.

Two Broad Subgroups of Siglecs

While Siglecs -1, -2, and -4 appear to be evolutionarily

rather conserved, the CD33/Siglec-3-related subgroup

(Human Siglecs -3 and -5 to -11) appear to be rapidly

evolving. Some CD33/Siglec-3-related Siglecs appear to

have evolved as hybrids of pre-existing genes and/or by

gene conversion. For these reasons, sequence compari-

sons alone do not allow the conclusive designation of the

orthologue status of all mouse genes, and additional

features such as gene position and exon structure must

be taken into account. Until such issues are resolved,

some mouse Siglecs have been assigned a temporary

alphabetical designation.

Common Structural Features

All are single-pass Type 1 integral membrane proteins

with extra-cellular domains consisting of uniquely

similar N-terminal V-set Ig domains, followed by

variable numbers of C2-set Ig domains, ranging from

16 in Sn/Siglec-1 to 1 in CD33/Siglec-3. Crystal

structures for mouse Siglec-1 and human Siglec-7

indicate that the V-set immunoglobulin-like fold has

several unusual features, including an intra-beta sheet

disulphide and a splitting of the standard beta strand G

into two shorter strands. These features along with

certain key amino acid residues appear to be require-

ments for Sia recognition. In particular, a conserved

arginine residue is involved in a salt bridge with the

carboxylate of Sia in all instances studied to date.

Cell-Type Speciﬁc Expression

With the exception of MAG/Siglec-4 and Siglec-6,

expression appears to be conﬁned to the hematopoeitic

and immune systems. Within these systems each Siglec is

expressed in a cell-type speciﬁc fashion, suggesting that

each may be involved in discrete functions. However,

systematic studies of Siglec expression outside the

hematopoeitic system and during development have

not yet been done.

Genomic Organization

and Phylogeny

Based on probing for the canonical functional amino

acids in the V-set domain of the typical Siglec, there is no

evidence for Siglec-like molecules in prokaryotes, fungi

or plants, nor in animals of the Protostome lineage,

including organisms for which the complete genome is

available. In contrast, it is relatively easy to ﬁnd Siglec-

like V-set domains in many vertebrate taxa (Sia

recognition by ﬁsh and reptile Siglec candidates has

not been formally shown as yet). While the relatively

conserved Siglecs (-1, -2, and -4) have clear-cut single

orthologues that are easy to identify in various species,

the remaining “CD33/Siglec-3 related” Siglecs appear to

have been evolving rapidly. Most of the latter genes are

clustered together in a , 500 kb region on human

chromosome 19q13.3– 13.4.

Siglec Recognition of Sialic Acids

and Their Linkages

The ﬁrst two Siglecs discovered (Sn/Siglec-1 and

CD22/Siglec-2) had strikingly different binding proper-

ties for sialosides—with Sn preferring alpha2–3 linked

targets and CD22 being highly speciﬁc for alpha2–6

linkages. In the latter case, the binding afﬁnity was in

the low micromolar range. MAG/Siglec-4 also has an

extended binding site that is even highly speciﬁc for the

underlying sugar chain. There is also variable pre-

ference for certain types of sialic acids, with Sn and

MAG not tolerating the common N-glycolyl modiﬁ-

cation of Sias. However the CD33/Siglec-3-related

Siglecs are more promiscuous in their preferences for

different types and linkages of Sias. Of course, many of

the less common linkages and types of sialic acids have

not been studied for Siglec recognition. The Golgi

enzymes that are potential regulators of Siglec func-

tions are primarily the sialyltransferases, and to some

extent the enzymes which modify sialic acids. Some

Siglecs show preferences for certain macromolecular

ligands e.g., CD45 for CD22/Siglec-2, the mucins

CD43, and Muc-1 for Sn/Siglec-1, and certain brain

glycolipids for MAG/Siglec-4.

Potential Effects of Neu5Gc Loss

on Human Siglec Biology

The most common Sias of mammalian cells are

N-acetylneuraminic acid (Neu5Ac) and N-glycolylneur-

aminic acid (Neu5Gc). Humans are an exception,

because of a mutation in CMP-sialic acid hydroxylase,

which occurred after the time (, 5–7 Ma) when we

shared a common ancestor with great apes. The

resulting loss of Neu5Gc and increase in Neu5Ac in

humans could have potentially altered the biology of the

Siglecs. For example, human cells have a higher density

of Sn/Siglec-1 ligands than great apes, the distribution of

Sn-positive macrophages in humans is different, and a

much larger fraction of human macrophages is positive.

Other emerging evidence suggests that there are further

human-speciﬁc changes in Siglec biology that may be

related to the loss of Neu5Gc.

Masking and Unmasking of Siglecs

Binding Sites on Cell Surfaces

The initial assumption was that Siglecs were involved in

intercellular adhesion. However, in most instances, their

binding sites appear to be masked by Sias on the same

cell surfaces on which they are expressed. Of course,

external ligands with very high afﬁnity/avidity may still

compete for the endogenous masking ligands. There is

also some evidence that unmasking can occur under

certain conditions, but it is not known if this is

biologically relevant. Overall, the signiﬁcance of Siglec

masking is unclear at this time.

Signaling Motifs in Cytosolic Tails

The CD33-related Siglecs have conserved tyrosine

residues in the cytosolic tails, one of which corresponds

to a canonical immunoreceptor tyrosine-based inhi-

bition motif (ITIM). Various in vitro manipulations of

these receptors indicate that these tyrosines are indeed

targets for phosphorylation, and that they can modulate

signaling events by recruiting certain tyrosine phospha-

tases. However, the true in vivo biological functions of

these signaling motifs remain obscure. Another major

unresolved question is: what is the connection between

extra-cellular sialic acid recognition and signaling via

the cytosolic motifs?

Known and Putative Functions

of the Siglecs

Various lines of evidence indicate that MAG/Siglec-4 is

involved in the maintenance of myelin organization

SIGLECS 39

and in the inhibition of neurite outgrowth during

regeneration after injury. It is also reasonably clear

that CD22/Siglec-2 functions as an inhibitory com-

ponent of the antigen receptor complex of B Cells, and is

thus involved in regulating the humoral immune

response. While Sn/Siglec-1 appears to mediate various

macrophage adhesion events in vitro and in vivo,itisas

yet unclear what the functions of these interactions are.

Little is known about the functions of CD33-related

Siglecs. It has been suggested that these molecules are

involved in innate immunity. One hypothesis currently

being tested is that Siglecs may be sensors for pathogens

that have sialylated cell surfaces and/or express extra

cellular sialidases.