Receptors as Drug Targets
Receptors are typically envisaged as cell
surface recognition sites for endogenous hor-
mones, neurotransmitters, and neuromodu-
lators. They are coupled to various signal
transduction systems located both within the
membrane and intracellularly, and can there-
fore regulate responses to the cellular/tissue
microenvironment. Compounds that bind to a
receptor are known as ligands. These may be
conceptualized as “keys” that fit into a specific
“lock” on the cell surface, the latter being the
receptor, to modify cellular activity at the bio-
physical, biochemical, and/or genomic level.
While major advances have been made
with respect to understanding the structure and
function of receptors, the “lock and key” hy-
pothesis proposed by Ehrlich and Langley over
a century ago remains the conceptual molec-
ular model for understanding the etiology of
human disease states, for defining drug action,
and for targeting and designing new therapeu-
tic agents.
Receptors can be defined in terms of their
selectivity, the saturability and reversibility of
ligand binding, and functionality. The defini-
tion of a receptor in both pharmacological and
physiological terms requires that it has spe-
cific interactions with ligands that belong to
a given pharmacological class. Thus for an
adrenergic receptor, it is important that the re-
ceptor recognize with high affinity (i.e., low
dissociation constant, in the nanomolar to mi-
cromolar range) agonists such as epinephrine
and norepinephrine as well as antagonists such
as phenoxybenzamine (α-adrenoceptor) and
propranolol (β-adrenoceptor). In contrast, an
adrenoceptor should show minimal affinity for
other ligand classes (e.g., selective calcium en-
try blockers, angiotensin converting enzyme—
ACE— inhibitors, and glutamate receptor lig-
ands). This provides a structure-activity rela-
tionship (SAR) for the interaction of a receptor
with its ligands.
The ability of a receptor to distinguish be-
tween enantiomers of a selective ligand, its
stereoselectivity, is a critical feature of a recep-
tor versus a ligand-binding site; the latter lacks
the ability to initiate a functional response.
However, not all receptors discriminate be-
tween enantiomers of all ligands. For in-
stance, while the α4β2 subtype of the nicotinic
cholinergic receptor labeled by [3 H]cytisine
clearly distinguishes between the R- and
Contributed by Michael Williams and Rita Raddatz
Current Protocols in Pharmacology (2006) 1.1.1-1.1.18
Copyright

C 2006 by John Wiley & Sons, Inc.
UNIT 1.
S- enantiomers of nicotine, it has no stereo-
selectivity for the enantiomers of the novel
nicotinic analgesic, epibatidine. The reasons
for this are unknown.
The saturability of a receptor relates to the
ability of a ligand to fully occupy a finite recep-
tor population in a tissue. A lack of saturabil-
ity reflected in a radioligand binding assay can
indicate the presence of low-affinity, “nonspe-
cific” sites that are not receptors. Ligand bind-
ing to a receptor should also be reversible,
reflecting the dynamic nature of a chemical
transmission process that reaches equilibrium
when the ligand association rate is equal to the
dissociation rate. Reversible binding is not al-
ways easy to demonstrate in vitro, especially
in the case of peptide ligands. A physiologi-
cally relevant receptor should also be shown
to mediate a functional response that can be
blocked by a selective antagonist.
The term “receptor” is now used in a
broader sense to describe any recognition site
for a drug-like compound. Thus, enzymes, up-
take sites, ligand-binding proteins (e.g., CRF
and acetylcholine-binding proteins), voltage-
gated ion channels, and intracellular targets
(e.g., NFκB, bcl2 ) are also considered as re-
ceptors along with the more traditional recep-
tors: ligand-gated ion channels (LGICs) and
G protein-coupled receptors (GPCRs). Strictly
speaking, the term “drug” describes a chemi-
cal substance that alters tissue function for the
benefit of organism function. The term drug
can also be used colloquially to describe recre-
ational substances like cocaine, amphetamine,
marijuana, and to a lesser extent, caffeine,
alcohol, and nicotine, substances that alter
perceptional and/or attentional states and can
cause habituation or addiction. These sub-
stances also produce their effects by acting at
receptors, either directly, e.g., marijuana in-
teracting with cannabinoid receptors, or in-
directly, as with cocaine-induced stimulation
of dopamine receptors by blocking dopamine
uptake into nerve terminals. Chemical sub-
stances used as research tools to define bi-
ological systems are termed “new chemical
entities” (NCEs), “compounds,” or simply lig-
ands, rather than as drugs. The latter term is
reserved for those NCEs active in humans.
The manner in which NCEs specifically
affect cellular function to produce beneficial
effects is an area of evolving knowledge. The
Receptor
Binding
1.1.1
Supplement 32
 majority of drugs in use today, many of which
were identified before their molecular targets
were identified, are receptor antagonists or en-
zyme inhibitors. This suggests that many dis-
eases result from excess receptor activity or
enzyme activation. Indeed, recent findings
suggest that receptors are not static entities but
rather may be constitutively active, i.e., the re-
ceptor may be activated even in the absence
of endogenous substances that stimulate the
recognition site. The concept of constitutively
active receptors (Kenakin, 1996; UNIT 9.5) sug-
gests there may be naturally occurring regula-
tors of intrinsic receptor function (i.e., inverse
agonists) whose malfunction or absence may
contribute to the etiology of some diseases.
Receptors are complex proteins with mul-
tiple potential ligand recognition sites, includ-
ing sites that may be distinct from the endoge-
nous agonist recognition site and may actually
reside on distinct proteins that are part of the
receptor complex. Such receptor modulatory
sites may represent novel drug targets, e.g.,
allosteric or modulatory sites (UNIT 1.21). The
effect of benzodiazepines (BZs) on GABAA
receptor function illustrates the conceptualiza-
tion of ancillary drug targets and the elusive
nature of the proposed endogenous modula-
tor, presumed to be a “BZ-like” substance.
The dynamic nature of the molecular events
underlying a variety of disorders, such as
bipolar illness and septic shock, can be il-
lustrated in the sequential activation, ampli-
fication, and induction of multiple modulator-
mediated events. In septic shock, the induction
of a toxic cytokine receptor-mediated cascade
has significantly complicated the search for
new drugs to treat this condition. This empha-
sizes the need to define key targets in critical
pathways rather than attempt to treat their se-
quelae. It is possible that many diseases are the
result of multifactorial events that vary during
the pathophysiological course of the illness.
For instance, >32 discrete gene loci have been
associated with schizophrenia. Therefore, tar-
gets that are downstream from key points in
the disease transduction pathway may not be
the optimal targets for treating the disorder.
RECEPTOR CLASSIFICATION
AND NOMENCLATURE
New receptors are identified on a regular
basis as scientists screen the various genomic
databases for novel sequences. These recep-
tors are frequently designated in an apparently
haphazard manner, often leading to confusion
Receptors as
Drug Targets as identical receptors are given different names
1.1.2
Supplement 32
by different groups. Kenakin (1993) recom-
mended receptor parsimony as an approach to
receptor classification, since in his view, “a
new receptor subtype should not be invoked
until absolutely necessary.” Despite the prag-
matism of this approach, receptor nomencla-
ture remains a challenge.
In an attempt to bring order to this pro-
cess, the International Union of Pharmacology
(IUPHAR) established a Receptor Nomen-
clature Committee with standing subcommit-
tees assigned for each receptor superfamily.
As data become available, these subcommit-
tees make recommendations for developing
a systematic nomenclature for each receptor
family. These recommendations are published
in Pharmacological Reviews and are collated
in the Sigma-RBI Handbook (see Internet
Resources), which is a valuable and definitive
online source.
RECEPTOR STRUCTURE
AND MOTIFS
Receptors are large macromolecules com-
posed of proteins, lipids, and carbohydrates
with molecular weights that vary from 30
to >300 kDa. Receptor proteins also un-
dergo posttranslational modification, includ-
ing phosphorylation, prenylation, glycosyla-
tion, palmitoylation, sulfation, and dimeriza-
tion, that contributes to their distinct functional
and pharmacological properties. Receptors are
currently divided into four major classes.
G Protein–Coupled Receptors
(GPCRs)
GPCRs are structurally represented by
a motif of seven hydrophobic membrane-
spanning amino acid helices (Fig. 1.1.1A),
each of which is between 20 and 30 amino
acids in length. This motif is used to describe
this group of proteins as seven-transmembrane
(7TMs or R7 G) receptors (Kenakin, 1996;
Strosberg, 1996). The complex signaling path-
ways modulated by GPCRs offer a variety
of potential therapeutic targets (Liebmann,
2004). GPCRs are coupled to various mem-
bers of the G protein superfamily, so named
because of their functional dependence on
the hydrolysis of the purine nucleotide, GTP,
for activity. The functional unit of GPCRs is
the receptor/G protein/effector. The effector
is one of a large family of intracellular pro-
teins that include adenylyl cyclase, the MAP
(mitogen-activated protein) kinases, phospho-
lipases A2 , C, and D, phosphatidylinositol-3
kinase (PI3K), pp60Src , p21, K+ and Ca2+
Current Protocols in Pharmacology
Figure 1.1.1 Structural motifs of various receptor classes. (A) GPCR with seven membrane-
spanning regions; (B-E) LGICs: (B) glutamate receptor, (C) P2X receptor, (D) nAChR, and (E)
VGIC K+ -rectified inward (Kirs) receptor; (F) STAT receptor; (G) PTK growth factor receptor; (H)
neutrophin receptor (trk).
channels, and other signaling proteins. Al-
though most cellular responses following acti-
vation of a GPCR are thought to be mediated
by G proteins, a growing number of GPCR-
mediated effects, such as src kinase activation,
have been shown to be G protein–independent,
suggesting this family of receptors would more
accurately be termed 7-TM receptors (Hall
et al., 1999).
The subunits of the G protein superfamily
are comprised of three heterologous subunits,
α, β, and γ. Eighteen distinct Gα, five Gβ,
and twelve Gγ isoforms have been identified
to date. The α subunits are divided into four
subgroups based on their major effector in-
teractions: Gs , which stimulate adenylyl cy-
clase; Gi/o , which are inactivated by pertus-
sis toxin (PTX) and inhibit adenylyl cyclase;
Gq/11 , which are PTX-insensitive and mediate
phospholipase C activation, and G12/13 , which
mediate cellular responses via Rho- and Rac-
GEFs (guanine nucleotide exchange factors).
The Gβγ subunits also regulate effector func-
tion, including βARK-1, PLA2 , adenylyl cy-
clase types II and IV, N-type Ca2+ channels,
and K+ channels. The heterotrimeric G pro-
teins are inactive when bound to GDP, and
Current Protocols in Pharmacology
GDP/GTP exchange is catalyzed by interac-
tion with activated GPCRs. The active G pro-
tein dissociates into α and βγ subunits that in-
teract with effector molecules and are rapidly
inactivated by the intrinsic GTPase activity
of the α subunit (responsible for hydrolyzing
GTP back to GDP) and the resulting reconsti-
tution of the heterotrimeric Gα, β, γ protein
complex.
The lengths of the extracellular and in-
tracellular loops that connect the transmem-
brane helices vary between different GPCRs,
including those that are members of the same
receptor superfamily. The amino acid com-
position of these loops determines the na-
ture of the ligand-binding site and aids in
defining the interactions of a given receptor
with the various G proteins. The second and
third intracellular domains and the C-terminal
tail confer G protein coupling specificity.
GPCRs can also be grouped into subfami-
lies that include the rhodopsin/adrenoceptor,
secretin/VIP, metabotropic glutamate, and
protease-activatable families, among others.
The endogenous ligands for these include
Receptor
small molecules, lipids, metals, peptides, and, Binding
in the case of rhodopsin, light.
1.1.3
Supplement 32
 Ligand-Gated and Voltage-Gated Ion
Channel (ICs)
ICs are composed of several distinct pro-
tein subunits. These are usually in the form
of a tetramer or pentamer that constitutes a
functional ion channel through which conduc-
tion is modulated by various ligands (Catterall,
2000). The ICs, unlike GPCRs, mediate fast
responses in cells. There are numerous ligand-
binding sites that contribute to the modulation
of channel function located on and between the
component subunits. Some of these sites are
in the pore. Strictly speaking, only the ligand-
gated ion channels (LGICs) can be considered
to be receptors, since they bind conventional
receptor ligands. Voltage-gated channels can,
however, be included in this family because
of the broader definition of receptors as lig-
and targets. Such compounds can modulate
the function of voltage-gated channels (e.g.,
charybdotoxin).
The ICs include several functionally char-
acterized receptor families that have differ-
ent structural motifs (Fig. 1.1.1B-E). LGICs
include GABAA , glutamate, serotonin-3 (5-
HT3 ), P2X, and nicotinic cholinergic (nAChR)
receptors. VGICs include K+ -rectified inward
(Kir’s) and K+ -rectified outward (Kv) ion
channels, calcium-activated potassium chan-
nels (KCa ), as well as Na+ -, Cl− -, Ca2+ -, cyclic
nucleotide-, and ATP-gated ion channels.
The ICs are very complex proteins having
many splice variants of different subunits that,
in turn, vary in composition. In the LGIC fam-
ily, each subunit of the channel can have ei-
ther two (e.g., P2X) or four (e.g., nAChR) hy-
drophobic membrane-spanning domains (Fig.
1.1.1C and D), termed M1 to M4. M2, which
forms the lining of the pore, is conserved
throughout all known LGICs and is respon-
sible for controlling ion selectivity.
The ligand specificity of LGICs is deter-
mined by the nature of the component sub-
units. For example, the binding site for nico-
tine on neuronal α4β2 nAChRs is formed by,
and between, two subunits. Homomeric ion
channels, those comprised of multiples of a
single type of subunit, are generally consid-
ered to be exceptions in nature, with heterolo-
gous ion channels like the α4β2 nAChR being
the norm. For many ion channel receptors, the
number of possible combinations and permu-
tations of constituent subunits and associated
proteins is in the thousands. At present, it is
unknown which represent naturally occurring
functional receptors.
Receptors as LGICs are modulated by a series of dis-
Drug Targets
tinct ligands, each interacting with its own
1.1.4
Supplement 32
discrete recognition site on one of the pro-
teins associated with the channel. In the case
of the GABAA receptor, a major modulator
site is represented by the BZ receptor. For the
N-methyl-D-aspartate (NMDA) subtype of the
glutamate receptor family, component sites in-
clude receptors for glycine, MK-801, and var-
ious polyamines.
VGICs are also multimeric protein com-
plexes that vary in composition between the
major families. Voltage-gated potassium chan-
nels comprise twelve families, Kv1 to Kv12,
and have six putative membrane-spanning
domains, termed S1 to S6, in an α sub-
unit, plus an associated β subunit that is
not membrane-spanning (Grissmer, 1997).
Voltage-gated sodium channels have an α sub-
unit with four homologous domains I through
IV, each of which has six putative membrane-
spanning regions and an associated β subunit.
Voltage-gated calcium channels consist of a
large α1 subunit that incorporates the pore,
the voltage sensor, and sites of known ligand
interactions plus three or four additional sub-
units that help modulate expression and elec-
trophysiological properties of the channel. The
ten identified α1 subunits have been classi-
fied into three major types; Cav1-3 and demon-
strate a similar topography to the VG sodium
channel α subunits, with four homologous do-
mains each containing six putative membrane-
spanning regions.
While the structural motifs of the various
classes of mammalian ICs vary considerably
(Fig. 1.1.1B-E), some show a high degree of
homology with gene products found in lower
organisms such as C. elegans and Drosophila
melanogaster. These include the ced family
of apoptotic genes. Defining the function of
proteins encoded by similar genes in simpler
organisms is often a useful strategy for estab-
lishing the function of the mammalian gene
products.
Transcription Factor Receptors
The superfamily of transcription factor re-
ceptors (also called nuclear hormone recep-
tors) includes intracellular ligand-dependent
transcription factors that regulate gene expres-
sion upon binding of their cognate ligands. The
steroid hormone receptors are the best char-
acterized members of the transcription factor
receptor superfamily and include the gluco-
corticoid (GR), progesterone (PR), estrogen
(ER), mineralcorticoid (MR), and androgen
(AR) receptors. Both agonists, e.g., estrogen,
and antagonists, e.g., tamoxifen, of these re-
ceptors are used clinically. Also included in the
Current Protocols in Pharmacology
transcription factor receptor superfamily are
the thyroid hormone (TR), vitamin D3 (VDR),
retinoic acid (RAR), retinoid x (RXR), peroxi-
some proliferators–activated receptor (PPAR),
ecdysone (EcR) receptors, as well as a number
of orphan receptors with structural homology
to the family. Many members of this superfam-
ily heterodimerize with a RXR and the com-
plex then binds to specific response elements
within the DNA promoter regions.
In addition, other families of transcription
factors, such as the signal transducer and ac-
tivator of transcription (STAT) proteins (Ihle,
1996), may be considered as receptors in that
they may be targets for bioactive ligands. The
STATs are latent cytoplasmic transcription fac-
tors that are structurally defined by a phos-
photyrosine binding domain, the SRC homol-
ogy 2 (SH2), which is required for STAT
dimerization and for subsequent interactions
with the JAK (Janus protein-tyrosine kinases)
family. This family of kinases phosphorylates
STATs and leads to activation of the JAK-
STAT signaling pathway. This family also in-
cludes other signal transduction elements like
the low-molecular-weight G protein family,
the transcription factors AP-1, NFκB, and NF-
AT, p48, the SMAD family of tumor suppres-
sor proteins, CSF-1 and the Bcl-2 and p53
families. These signaling elements are typi-
cally activated downstream of other receptors,
including GPCRs and tyrosine kinase recep-
tors. The hormone response elements (HREs)
on DNA themselves may also be targeted by
ligands to block interaction with transcrip-
tion factors and thereby block subsequent gene
transcription.
Enzyme-Associated Receptors
The enzyme-associated receptor superfam-
ily (including growth factor receptors) con-
sists of single- or multi-subunit proteins that
contain a subunit with a single transmem-
brane domain. The largest groups within this
superfamily are the protein-tyrosine kinase
(PTK) receptors (Fig. 1.1.1G) that include
PDGF, EGR, FGF, IGF, HGF, VEGF, and neu-
rotrophin (trk; Fig. 1.1.1H), and contain ki-
nase domains within their protein structure.
Also included in the superfamily are the mul-
timeric complexes that utilize kinases, such as
the JAK-type kinases, for their signal trans-
duction. This family includes the Class I and
Class II cytokine receptor families; the tumor
necrosis factor (TNF) receptor family; anti-
gen receptors (TCR, BCR); and the type II
serine/threonine kinase receptor family that
Current Protocols in Pharmacology
includes TGFβR (transforming growth factor-
β) and ActR. Members of the Class I cy-
tokine family are characterized by the pres-
ence of multiple fibronectin type III–like mo-
tifs and include receptors for the interleukins
IL-2, IL-3, IL-5, IL-6, IL-7, IL-9, and IL-11;
EPO, prolactin, and ciliary neurotrophic fac-
tor (CNTF); and granulocyte/macrophage and
granulocyte colony-stimulating factors (GM-
CSF and G-CSF). The Class II cytokine recep-
tor family includes IL-10R and the interferon
receptors IFN-α/βR, IFN-γ-Rα, and IFN-
γ Rβ. The receptors pp60src and p56lck rep-
resent two classes of tyrosine kinase that are
not activated by traditional receptor ligands.
The delineation of functions of ICs,
GPCRs, transcription factor receptors, and
enzyme-associated receptor families is not
always clear. Cytokines can activate STATs
(Leaman et al., 1996), as can the GPCR for
angiotensin II. GPCRs also modulate channel
function, while some LGICs produce their ef-
fects in association with G protein–coupled
receptor systems. Because of this, the receptor
motif remains the most critical element in as-
signing a receptor to a particular superfamily,
and the associated signal transduction mecha-
nism is secondary.
RECEPTOR LIGANDS
Compounds that interact with receptors are
characterized in terms of two basic properties:
affinity and efficacy (Kenakin, 1996; Kenakin
and Onaran, 2002). The affinity of a com-
pound is described in terms of its dissociation
constant (Kd ) with respect to the receptor. Ac-
cordingly, affinity describes the strength of the
attraction between the receptor and the ligand.
The units for the dissociation constant, derived
from the Law of Mass Action, are molar (M). A
typical neurotransmitter or hormone-receptor
ligand will have a Kd value in the range of 0.1
to 10 nM. The lower the Kd value, the higher
the affinity of the ligand for the receptor.
Efficacy describes the capacity of a ligand
to elicit a biological response. Those ligands
that produce a positive response, such as stim-
ulation of adenylyl cyclase or activation of
an ion channel, mimic the actions of the en-
dogenous ligand for the receptor and are said
to have positive efficacy and, therefore, are
known as agonists. By definition, a full agonist
is a ligand that produces the same maximal ef-
fect (100% or unity) as that observed with the
endogenous ligand in a given functional sys-
tem. Intrinsic activity (IA) is a proportionality
Receptor
factor introduced by Ariens (1954) to describe Binding
1.1.5
Supplement 32
 Figure 1.1.2 Ligand efficacy spectrum.
the ability of a ligand to elicit a response in
relation to the maximal observable response
in a given tissue. The concept of IA assumes
a linear relationship between receptor occu-
pancy and tissue response, and is a function of
both the efficacy and the affinity of a ligand.
In practical use, IA is used to define relative
maximal responses when comparing a series
of ligands in a particular tissue. Intrinsic effi-
cacy of a ligand is a proportionality factor that
defines the stimulus per receptor molecule that
is produced by an agonist. Recently, agonists
have been identified with intrinsic activities
(IA) of >100%. While these were initially
termed “super agonists,” such agents are, in
reality, full agonists. The lesser activity of the
standard full agonist reflects the fact that it is
either a partial agonist or causes a rapid desen-
sitization of the receptor. In many instances,
the relationship between the stimulus and an
agonist response is nonlinear, indicating that
the receptor/transduction process can amplify
receptor stimulation.
Compounds that bind to the receptor that
have no effect on their own but which block
the actions of endogenous and exogenous ago-
nists have zero efficacy and are termed neutral
antagonists. Compounds with effects opposite
to those of an agonist display negative effi-
cacy and are known as inverse agonists or pos-
itive antagonists. This type of compound has
an efficacy value of −1. Thus, the spectrum of
ligand activity can, by definition, range from
Receptors as −1 to +1 (Fig. 1.1.2). Accordingly, the effects
Drug Targets
1.1.6
Supplement 32
of an inverse agonist are blocked by a neutral
antagonist.
Ligand efficacy is a complex, poorly under-
stood, and actively debated concept, especially
in the context of the in vivo effects of drugs.
Efficacy encompasses a number of physio-
chemical factors including affinity, tissue (i.e.,
receptor) access (distribution), promiscuous
receptor coupling to different transduction
mechanisms, metabolism, and plasma protein
binding, among others. The concept has be-
come increasingly complex as more is learned
about the molecular aspects of receptor activa-
tion and signal transduction, requiring an in-
tegrative, iterative systems approach for both
receptor and ligand characterization. Thus, lig-
and binding and functional data derived from
a transfected human receptor system must be
compared with data obtained with the receptor
in its native, “wild” state and in different tissue
systems.
CONSTITUTIVELY ACTIVE
RECEPTORS
An important new concept is that of con-
stitutively active receptors (UNIT 9.5). These
are receptors, currently confined to GPCRs,
which are active in the absence of a ligand.
Conceptually, it is thought that G protein–
activating sections of the 7TM receptor struc-
ture are in contact with the relevant G pro-
tein, leading to signal transduction. Ligand
efficacy has therefore been redefined as “the
Current Protocols in Pharmacology
property of a [compound] that modifies sub-
sequent interaction of the 7TM receptor with
other membrane proteins” (Kenakin, 1993).
Furthermore, Kenakin (1996) has postulated
that the inactive form of a GPCR is a spe-
cialized case. This raises the possibility of
endogenous inhibitory factors that modu-
late constitutive receptor activity. Indeed, the
agouti-related protein (AgRP) is released from
neuronal terminals and acts as both an inhibitor
of α-MSH-induced activity and as an endoge-
nous inverse agonist of melanocortin MC(3)
and MC(4) receptors (Adan and Kas, 2003).
Additionally, accessory proteins could modu-
late the interaction between the receptor and
the G protein and therefore may represent im-
portant new drug targets. Evidence for this
concept comes from work with PD 81,723,
a putative allosteric modulator of the adeno-
sine A1 GPCR (Kollias-Baker et al., 1997). In
addition to effects enhancing the binding and
actions of the endogenous ligand, adenosine,
PD 81,723 appears to increase constitutive re-
ceptor activity.
Constitutive receptor activity adds yet an-
other layer of complexity to the characteriza-
tion of receptors and ligands. The potential
existence of endogenous modulators of con-
stitutively active receptor function may make
redundant the theoretical concept of spare re-
ceptors per se, as the latter may simply reflect
the degree of endogenous modulation of the
receptor-G protein interaction in a given tissue.
The assumption that the receptor (and en-
zyme) population of a given tissue system
is static is no longer a viable concept. The
levels of both receptors and enzymes can be
altered (Donaldson et al., 1997) as a result
of trauma (e.g., hypoxic, ischemic, or physi-
cal damage), disease (e.g., inflammation, vi-
ral, or bacterial infection), or development
(Clifford et al., 1997). The concept of disease-
related, inducible receptors and enzymes adds
yet another layer of complexity and oppor-
tunity to the drug discovery process. Viruses
have been identified that activate dormant
genes in host cells that express constitutive
chemokine receptors or promote incorpora-
tion of the virus genome into the host cell
genome (Arvanitakis et al., 1997). The abil-
ity of human cytomegalovirus (CMV) to en-
code a β-chemokine receptor related to the hu-
man chemokine receptors CCR5 and CXCR4
as a means to facilitate HIV entry provides
a pathophysiological rationale for the effect
of viruses on host cell chemokine expression
(Pleskoff et al., 1997). Similarly, the finding
Current Protocols in Pharmacology
that chronic pain can induce spinal cord no-
ciceptive neurons to express the algesic neu-
rokinin substance P (Neumann et al., 1996)
represents another major conceptual advance
in understanding receptor function in the con-
text of pain processing.
Some drugs have been in use for many years
and served as ligands to define receptor fami-
lies long before endogenous agonists for those
receptors were discovered. An example of this
is the opiate receptor family. While morphine
and related agents were known to produce their
analgesic effects by interacting with receptors,
it was not until the 1970s, with the identifi-
cation of the enkephalins and endorphins as
endogenous ligands for these sites that a bona
fide target was identified. More recently, the
tetrapeptide endomorphins have been found to
be potent agonists for the µ opiate receptor
(Zadina et al., 1997). Similar efforts targeted
towards identifying the endogenous ligand for
cannabinoid receptors resulted in the identifi-
cation of anandamide (Devane et al., 1992).
In a diseased or traumatized tissue,
receptor-mediated responses are frequently
modified to the detriment of the organism, as
noted above in the case of viral infections.
Examples include constitutive β-adrenoceptor
activation in hypertension, decreased insulin
production in diabetes, and hyperactivation of
glutamate receptors during stroke-related hy-
poxia and ischemia. A drug may be used to
restore normal function by mimicking the ef-
fects of an endogenous ligand or by blocking
the actions of an endogenous agonist. It is also
possible that while the level of the endogenous
ligand is normal, the number or sensitivity of
receptors is increased. An antagonist may be
of therapeutic benefit in such circumstances
reflecting a diminution in constitutive modu-
lator activity. To better define the pathophys-
iology of human disease states at the molec-
ular level, new technologies must be applied,
including differential display in tissues from
in vitro and in vivo disease models and posi-
tional and functional cloning techniques using
human genomic databases.
LIGAND-RECEPTOR
INTERACTIONS
Ligands (L) are thought to interact with the
receptor (R) in a reversible, competitive, and
saturable manner in accordance with the Law
of Mass Action:
Equation 1.1.1 Receptor
Binding
1.1.7
Supplement 32
 The formation of an RL complex imparts to
the receptor an ability to alter cellular function
by interaction with transmembrane signaling
proteins or, in the case of ion channels, by a
change in ion flux. Receptor isolation, cloning,
and point mutation studies have confirmed that
the binding of a ligand to its receptor is an event
distinct from that of receptor coupling to the
second messenger systems. The RL complex
formation thus results in either an alteration
in the spatial relationship of the receptor and
the transmembrane signaling proteins or, alter-
natively, a change in the steric conformation
of the ligand, thermodynamically favoring the
transduction process.
For enzymes, the situation is similar to that
for RL complex formation, except that the
enzyme-substrate (ES) complex results in the
formation of a product as shown in the follow-
ing equation:
Equation 1.1.2
The substrate (S) undergoes a catalytic con-
version, whereas both the receptor and ligand
are unchanged at the end of the transduc-
tion process described by the first equation
above. With the ES complex, the reaction can
be driven backward by feedback inhibition of
excess product, depending upon the concentra-
tions of the various components at equilibrium.
For an analogous situation with a receptor-
mediated event, a receptor response can be
diminished by receptor downregulation (e.g.,
internalization or phosphorylation), a decrease
in receptor number, or a functional uncoupling
of the signal transduction mechanism.
While the Law of Mass Action may be
extrapolated from enzymes to receptors, the
use of Michaelis-Menten kinetics to describe
receptor behavior is an approximation rather
than a strict extrapolation of enzyme theory. As
discussed in UNITS 1.2, 1.3 & 2.1, receptors may
be studied in the context of their concentration-
or dose-dependent effects on radioligand bind-
ing, or in terms of a biochemical or physiolog-
ical tissue response.
Receptor-ligand (RL) interactions are de-
scribed in vitro by a number of terms. In
binding studies, the dissociation constant (Kd ),
a measure of receptor affinity, and receptor
density (Bmax ) are derived from a saturation
isotherm (Fig. 1.1.3A). In this type of ex-
periment, the receptor concentration is held
constant while the radioligand concentration
Receptors as is increased until the radioligand saturates
Drug Targets
all the specific binding sites on the receptor.
1.1.8
Supplement 32
Historically, binding data have been analyzed
using the Scatchard equation, which plots ra-
dioligand bound (B) versus B divided by the
amount of free radioligand (F). The Kd and
Bmax can be derived from the plot of B versus
B/F (Fig. 1.1.3B). However, the Scatchard plot
is limited in that B is present on both the or-
dinate and the abscissa, constraining the data
towards linearity. To overcome this limitation,
nonlinear regression analysis using computer
programs (e.g., GraphPad Prism, EBDA, or
Ligand) can be employed to analyze data de-
rived from binding site saturation analyses, to
assess whether the data fit a single- or multiple-
binding-site model, and to derive the corre-
sponding Kd and Bmax values. The Bmax should
always be expressed in terms of milligrams of
protein, not gram tissue weight, since the latter
can vary considerably depending on the indi-
vidual tissue preparation.
Concentration-response curves (Fig.
1.1.3C) are used to derive an IC50 value, the
concentration of an antagonist required to
inhibit 50% of the radioligand binding. Since
the IC50 value is a function of receptor affinity
and the concentration of radioligand used, it is
a relative value. A more absolute value is the
Ki , which is derived using the Cheng-Prusoff
equation: Ki = IC50 /(1 + c/Kd ), where c =
the concentration of radiolabeled ligand and
Kd = the affinity constant of the ligand for the
receptor. The Ki value corrects for differences
in the concentration of radioligand and the
receptor affinity. As a result, the affinity of
a compound in a series of different binding
assays can be appropriately compared.
The EC50 value is the concentration of an
agonist that produces a response that is half
the maximal response produced by a saturat-
ing concentration of the ligand (Fig. 1.1.3D).
The EC50 value for a full agonist (A) is half
the maximal response that can be observed
in the system. For a partial agonist (B), the
EC50 may be derived either as the EC50 for
half the maximal response achieved by ago-
nist B, or as the concentration of agonist B
that produces the same half-maximal response
as that found for agonist A. The difference be-
tween the two EC50 values reflects the intrin-
sic efficacy of each compound. When evaluat-
ing a weak partial agonist like C, the response
may never approach even half the response
observed with the full agonist.
The generation of concentration-response
curves in the absence or presence of an an-
tagonist is used to derive a pA2 value, which
is defined as the negative logarithm of the
molar concentration of an antagonist that
Current Protocols in Pharmacology
Figure 1.1.3 (A) Schematic of a saturation binding curve: total, nonspecific, and specific binding
(see UNIT 1.3). (B) Scatchard derivation of specific binding saturation isotherm. (C) Ligand dis-
placement curve showing IC50 relationship. (D) Ligand efficacy and EC50 derivation. The EC50 for
a partial agonist (EC50 B) can be determined as the concentration at which a similar response
to that of a full agonist (EC50 A) is observed. Alternatively, the EC50 for a partial agonist can be
determined as the concentration at which 50% of the maximal response to the partial agonist is
determined (EC50 C). Clearly, using the latter approach in the absence of any measure of lig-
and potency (receptor affinity) can provide misleading data. (E) Dose-response relationship in the
presence of increasing concentrations (X-Z ) of an antagonist. The antagonist produces a classical
dose-dependent rightward of the agonist response. (F) Schild derivation of the data in E to derive
a pA2 value (see UNIT 1.2).

produces a two-fold rightward shift of the
agonist concentration-response curve (Fig.
1.1.1E). This dose ratio (dr) represents the
increase in agonist concentration needed to
achieve a given response in the presence of the
antagonist. The pAx relationship was identified
by Schild in 1949 and is derived from a Schild
plot or Schild regression (Fig. 1.1.3F). The
pA2 is equivalent to the pKB , which is the neg-
ative logarithm of the equilibrium dissociation
constant for the antagonist-receptor complex.
ORPHAN RECEPTORS
As new receptors are cloned from genomic
databases (Hopkins and Groom, 2002). their
sequences are usually compared to those of
other known receptors to see whether they be-
long to an established family. Sequence ho-
mology within a family can vary between 25%
and 100%, and often the individual transmem-
brane (TM) sequences are used to identify ho-
mology. There are no absolute rules regard-
ing the degree of homology required to assign
a new protein to a particular receptor fam-
ily. By this process, a large number of or-
phan receptors have been identified in both
the GPCR and transcription factor receptor
families.
The identification of new proteins that be-
long to a particular receptor family on the ba-
sis of their motif, sequence homology, and in
some instances, effector mechanisms, has re-
sulted in the discovery of a number of orphan
receptors. These are defined by the absence of
any known, endogenous agonist ligand. One Receptor
of these is the opioid-like orphan receptor, Binding

1.1.9
Current Protocols in Pharmacology Supplement 32
 LC132. While this putative receptor shows se-
quence homology to the cloned µ-, δ-, and
κ-opioid receptors, no known opioid receptor
ligands interact with this site with high affin-
ity. A heptadecapeptide termed orphanin FQ
(OFQ), or nociceptin, was isolated from brain
that had high affinity (∼0.2 nM) for LC132.
Accordingly, the receptor was termed OFQ/N-
R or NOP (Ardati et al., 1997).
Although sequence homology can often
help categorize receptors into known families
and subtypes, changes in a single key amino
acid in receptors such as the 5-HT1B recep-
tor (Oksenberg et al., 1992), an ∼450-amino-
acid GPCR, can markedly affect the pharma-
cology of the rodent as compared to the hu-
man form of the receptor. The 5-HT1B receptor
was originally identified in the rat, but a hu-
man homolog was not readily found. Although
a human brain gene sharing 93% of the de-
duced protein sequence of the rat receptor was
identified, its pharmacology, as assessed by ra-
dioligand binding assays, was very different.
Thus, methysergide, which has a Ki value of
1823 nM at the rat 5-HT1B receptor, has a Ki
value of 130 nM at the human homolog of the
receptor. Similarly, (-)-propranol, which has a
Ki value of 57 nM at the rat 5-HT1B recep-
tor, has a Ki value of 8100 nM at the human
homolog. These discrepancies suggest that the
two receptors are clearly different from a phar-
macological standpoint. However, replacing
the threonine residue at amino acid 355 in the
human version of the 5-HT1B receptor with an
asparagine (T355N) to correspond with the rat
receptor made the human receptor identical to
the rat in terms of its pharmacological selectiv-
ity. The fact that a single amino acid, ∼0.2%
of the total protein in this GPCR, can cause
such a dramatic change in the pharmacology of
the receptor provides a cautionary note regard-
ing extrapolation of radioligand binding data
from laboratory animals to human receptors.
It also highlights the possibility of phenotypic
differences in receptor recognition site char-
acteristics even when the proteins are as much
as 93% identical. Thus, when receptor homol-
ogy is based on 50% to 70% shared identity,
there is a need for some caution in extrapolat-
ing pharmacological and signal transduction
properties from one homolog to another.
The identification of a new protein that be-
longs to a known receptor family represents
only the first step in its use in defining function
and disease etiology—a fact that is sometimes
lost in the excitement of cloning. Because a
Receptors as protein can be assigned to a family does not
Drug Targets
mean that its function is identical to that of
1.1.10
Supplement 32
other members of that group. In Alzheimer’s
research, two key proteins associated with fa-
milial forms of the disease, the presenilins 1
and 2, have structural motifs indicating mem-
bership in the 7TM family, although these are
intracellular proteins that exist in large pro-
tein complexes with aspartyl protease activity
(gamma secretases). Even though their struc-
ture has been known for several years, there is
still ongoing debate about their function and
importance as potential targets in Alzhemier’s
disease therapies. The transition from structure
to function is a major challenge for receptor re-
search that should not be underestimated, es-
pecially with the increased focus on the novel
proteins identified by genomics.
As previously discussed, endogenous ago-
nists for some orphan receptors may not have
been identified to date because their regula-
tion may occur via endogenous inverse ago-
nists, such as the agouti-related protein. There
is also the possibility that some of these 7TM
motif orphan receptors represent endogenous
modulators of constitutive receptor activity or
“decoy” proteins whose function is not that
of a typical ligand-recognition site, but rather
maintain tone in the signal transduction cas-
cade or act as G protein “sinks” that regu-
late the activity of other receptors in the cell.
Some orphans may remain un-liganded due to
a need for accessory proteins as was seen with
one previous orphan receptor that requires co-
expression of RAMP1 (receptor-activity mod-
ifying protein 1) to become a functional CGRP
receptor complex (Poyner et al., 2002). Recent
studies have demonstrated a novel role for the
GPCR Mas receptor as an antagonist of the
AT1 (angiotensin II type 1) receptor when they
form a heterodimer (Kostenis et al., 2005). The
complexity of functional roles receptors may
play in vivo makes identification of ligands
and functions for orphan receptors a complex
undertaking.
NEUROTRANSMITTERS,
NEUROHORMONES, AND
NEUROMODULATORS
Chemical communication between cells
and tissues has several hierarchical levels that
are defined on the basis of the temporal ef-
fects of the different natural effector classes
for the receptor (Bloom, 1988). Neurotrans-
mitters are released close to their target recep-
tors and cause rapid and specific effects that
result in a rapid depolarization or hyperpolar-
ization of the neuron. The consequences of
this action are integrated postsynaptically to
Current Protocols in Pharmacology
determine the spatial and temporal pheno-
type of subsequent neurotransmission pro-
cesses from the target neuron or cell. This may
result in the integration of the effects of two
or more neurotransmitters or, as in the case of
chronic pain input (Neumann et al., 1996), a
major change in the type of neurotransmitter
being produced in the target neuron.
Neurotransmitter effects are short lived,
typically in the millisecond timeframe. Neu-
romodulators, on the other hand, have a more
prolonged duration of action and serve to reg-
ulate the effects of neurotransmitters. While
neuromodulators are released in a manner sim-
ilar to neurotransmitters, they are not restricted
to the nerve terminal immediately proximal to
the target receptor. The GPCRs are involved
in many neuromodulatory processes since, in
addition to their effects on biochemical and
ion channel–related events, they can induce
immediate early genes, which may extend the
consequences of their actions by days or weeks
(Laduron, 1992). Neuropeptides are classical
neuromodulators.
Hormones such as growth hormone and in-
sulin are longer-acting entities, whose effects
are usually produced at a site distant from their
release. Their target site(s) are accessed via the
systemic circulation. Hormones are essential
for growth, development, reproduction, reg-
ulation of intermediary metabolism, and the
overall homeostasis of the organism. They pro-
duce their effects over periods of days to years.
Alterations in hormone levels and function re-
sult in puberty, menopause, and aging. De-
creases in estrogen levels have been associated
with the time to onset of Alzheimer’s disease
and breast cancer. Paracrine hormones are pep-
tides produced by endocrine glands and in the
intestine. Included in this group are gastrin and
somatostatin.
Autacoids are substances that are pro-
duced locally in response to tissue injury
and include serotonin, histamine, bradykinin,
the eicosanoids (prostaglandins, leukotrienes,
and thromboxanes), platelet-activating factor
(PAF), and lymphokines. The response to
these agents is rapid, although they have often
been described as local hormones. However,
since they do not use the systemic circulation
to reach their site of action, but rather act lo-
cally at the site of the inflammatory insult, they
are distinguished from hormones.
ALLOSTERIC LIGANDS
Allosteric ligands are compounds that indi-
rectly modulate receptor function by interact-
ing with sites on the receptor that are different
Current Protocols in Pharmacology
from those binding the endogenous ligands or
with sites on proteins associated with a recep-
tor. The concept of allosterism has evolved
from the classical model of Monod et al.
(1965) and the sequential model of Koshland
et al. (1966), which were derived from knowl-
edge of the subunit interactions of hemoglobin,
the enzyme pyruvate dehydrogenase, and
the Torpedo electroplax nicotinic cholinergic
receptor.
An established group of allosteric ligands
are the BZs that modulate GABAA recep-
tor function. These drugs are effective and
safe anxiolytics, anticonvulsants, and hyp-
notics. Because of the superior safety profile
of BZs compared to direct-acting receptor lig-
ands, it has been postulated that allosteric
modulators have an advantage over recep-
tor agonists and antagonists as drug candi-
dates. All types of LGICs have allosteric
modulators, and some share common recog-
nition sites for compounds like MK 801. It
is only recently, however, that allosteric mod-
ulators of GPCRs have been identified (May
et al., 2004). In retrospect, it is now appreci-
ated that the first of these were the various
cations and anions known to modulate lig-
and affinity at these receptors and their signal
transduction processes. Other types of ligands
found to disrupt G protein interactions with
GPCRs were mastoparan and the peptide ade-
noregulin. Moreover, compounds such as PD
81,723, which acts at the adenosine A1 recep-
tor, and obidoxime and alcuronium, which act
at m2 muscarinic receptors, are also selective
allosteric modulators. Allosteric modulators
often demonstrate subtype selectivity amongst
related GPCRs that has not been achieved for
ligands acting at the orthosteric site.
pH can also affect receptor function, which
may be an important allosteric effect related to
the pH changes involved in tissue trauma and
ischemia. In this context, nitric oxide (NO) se-
questered in hemoglobin (Hb) enhances deliv-
ery of oxygen to tissues where oxygen ten-
sion is low, since under this condition, Hb
releases NO to cause vasorelaxation and in-
crease Hb flow and oxygenation (Stamler et al.,
1997).
HUMAN RECOMBINANT
RECEPTORS
In the effort to understand disease etiol-
ogy and discover new drugs, the ultimate fo-
cus is on human receptors. Traditionally, the
study of receptor function was initiated with
the more easily accessible rodent (at either Receptor
Binding
the membrane or whole-animal level) or with
1.1.11
Supplement 32
 immortalized human cell lines that are usually
derived from tumors. While both approaches
have been extremely useful, they are limited in
that neither reflects the native, wild-type hu-
man receptors. Indeed, in some cases, it has
been found that these receptor sources do not
even approximate the wild-type human recep-
tor, leading to considerable complications in
the transition of compounds from preclinical
animal models to human trials. This has been
found with respect not only to efficacy and
selectivity, but also to safety.
The use of transfected human receptors in
cell lines that do not spontaneously express
the receptor of interest (“null” cell lines) has
been made possible by the ability to clone
human receptors. Human gene expression in
such cells has become a critical tool in recep-
tor research, although it is limited by a num-
ber of factors that are not always considered
when interpreting data from transfected cells
(Kenakin, 2003).
Ideally, when human DNA is incorporated
into the genome of a cell, the receptor is a per-
manent feature of the phenotype of the trans-
fected cell as it replicates (stable transfection).
In many instances, due to problems with the
vector or other experimental parameters, the
human receptor is only transiently expressed,
appearing as part of the cell phenotype for
a single or a limited number of cell cycles.
While this is better than no expression at all
and provides a means to assess receptor func-
tion using electrophysiological or biochemical
techniques, it creates problems in obtaining
reproducible data.
Because gene transfection and expression
are a function of the individual experiment, the
receptor may be underexpressed, normally ex-
pressed, or overexpressed in any given study.
Since the functional response to a ligand is
related to the ligand efficacy, receptor density,
and receptor interactions with endogenous sig-
nal transduction mechanisms, this variability
can lead to compounds being characterized as
partial or full agonists not because of their
intrinsic properties, but because of character-
istics of the experimental system. For exam-
ple, with overexpression of GPCRs, there is
a potential for the receptor to be promiscuous
and activate different members of the G pro-
tein superfamily that are not normally affected
by receptor activation. This effect may explain
differences in tissue and regional responses
to a given receptor ligand. Depending on the
array of signal transduction mechanisms and
Receptors as their relative abundances, the tissue response
Drug Targets
to a ligand may vary from full agonist to an
1.1.12
Supplement 32
inverse agonist. Under normal conditions, a
natural ligand may be capable of achieving se-
lectivity by virtue of its differing spectrum of
signaling efficacy in a number of distinct tis-
sue systems. Therefore, an exogenous ligand
may produce a different pharmacological pro-
file from the endogenous ligand (or known an-
tagonist) by activating (or inhibiting) systems
differently.
In developing a structure-activity relation-
ship (SAR) for a new series of chemicals, the
more that is known regarding the efficacy pro-
file and receptor selectivity of a ligand across
a number of tissue systems, the easier it is to
interpret the in vivo animal and human data
collected with these compounds. Much of the
tissue target selectivity of new compounds that
is ascribed to “pharmacokinetics” may reflect a
lack of understanding of the molecular proper-
ties of a compound in different tissue systems.
Cyproheptadine, widely used as a serotonin re-
ceptor antagonist, is also a potent antagonist of
histamine and acetylcholine receptors. Like-
wise, the serotonin uptake blocker fluoxetine,
one of the most widely used antidepressants,
has recently been found to block nicotinic re-
ceptors at therapeutic doses (Garcia-Colunga
et al., 1997).
As new compounds are advanced, receptor
binding profiles that include the assessment of
their activities at >80 GPCR, ion channel, en-
zyme, and uptake-site assays in vitro can pro-
vide important information about their value
as experimental tools and therapeutic agents.
A negative finding about a compound in one
of ten or fifteen assays should focus attention
on what parameters were being investigated in
the one assay rather than calling into question
the other nine or fourteen assays. Furthermore,
selectivity is a relative term, with many exam-
ples where in vitro activity does not have in
vivo consequences.
A further complication in ligand charac-
terization is related to the potential cell cycle
dependence of receptor coupling and the ef-
fects of the receptor environment on the ligand
response to any one component in the pres-
ence of others. This relates to both receptor
interactions or dimerization (e.g., dopamine
D1 and D2 receptors; adenosine A2A and
dopamine D2 receptors) and interactions be-
tween different signaling cascades. For exam-
ple, it is necessary to interpret with caution
data derived from the use of intact tissue sys-
tems where a cholinergic tone is either induced
or removed to enhance observation of the re-
sponse evoked by another class of receptor lig-
and. This underlines the need for an integrative
Current Protocols in Pharmacology
systems approach to ligand characterization.
In the absence of stably transfected cells that
have binding characteristics similar to the
wild-type human receptor, it is imperative that
interesting new ligands also be defined using
tissue systems containing the native receptor
whenever possible (Chapter 4).
An example of the caution required in inter-
preting data from transfected cell lines is the
report of a cloned P2 purinergic GPCR desig-
nated as P2Y7 (Akbar et al., 1996). While this
receptor was shown to be sensitive to ATP,
it was subsequently found to be identical in
primary structure to a leukotriene B4 (LTB4 )
receptor known as BLTR (Yokomizo et al.,
1997). Further examination of the Cos-7 cell
line used to identify the P2Y7 receptor showed
that it contained intrinsic purinergic receptors
and, in this system, the P2Y7 /BLTR receptor
responded to 1 µM ATP, an endogenous ligand
for the P2 receptor, and resulted in an increase
in intracellular calcium levels. However, when
BLTR was transfected into C6-15 glioma cells,
which have negligible levels of P2 purinergic
receptors, ATP had no effect on intracellular
calcium at concentrations up to 300 µM, while
10 nM LTB4 was fully efficacious.
Receptor characterization using a series of
ligands is dependent on a logical SAR that
provides information on the recognition site.
When a large number of diverse structures are
found to interact with a binding site, efforts
must be made to ensure that the site being stud-
ied is a biologically relevant receptor rather
than merely a radioligand binding site.
As new receptors are identified, the search
for novel ligands represents a major chal-
lenge. Black (1989) outlined his Nobel Prize-
winning work regarding β-adrenoceptor and
histamine H2 receptor blockers based on mod-
ifications of the natural agonist. However, this
approach is not always successful, and in many
instances years after a receptor has been iden-
tified the only known ligands are close struc-
tural analogs of the natural agonist. For over
a decade, antagonists for the tachykinin re-
ceptor family remained undiscovered until, in
the space of 6 to 9 months, high-throughput
screening efforts at Sanofi, Rhone Poulenc
Rorer, Sterling Drug, and Pfizer resulted in
a series of selective, nonpeptide, neurokinin
antagonists (Williams and Gordon, 1996).
For several systems, receptor classification
is based on the rank order potency of a lim-
ited series of agonists due to the paucity of
effective and selective antagonists. Receptor
characterization at the functional level is then
severely limited, especially when the avail-
Current Protocols in Pharmacology
able ligands lack bioavailability or appropriate
pharmacokinetics. While as many as 17 recep-
tors in the P2 purinergic receptor superfamily
have been identified by cloning, the pharma-
cological classification of these receptors is
based on a series of analogs of ATP and ADP
that may also function as ectonucleotidase in-
hibitors. Antagonists for the P2 receptor fam-
ily include a limited number of modified ATP
analogs, suramin (a compound that directly in-
teracts with G proteins) and a group of dyes.
Advances in the area of P2 receptors are lim-
ited, not by the techniques available or the di-
versity of the receptors identified, but by the
lack of suitable ligands that would permit char-
acterization and assignment of function to the
different molecular forms of the receptor.
RECEPTOR MUTATIONS
AND CHIMERAS
Receptor pharmacology has traditionally
involved defining the SAR of a series of
ligands or the relative activities of a number of
different pharmacophores. Since the tools of
molecular biology can be used to selectively
alter the amino acid composition of a protein,
it is possible to modify the amino acid
composition of the natural, wild-type receptor
to identify those constituents that are involved
in ligand binding and signal transduction.
As already noted, alteration of a single
amino acid in the human 5-HT1B receptor
radically alters its pharmacology (Oksenberg
et al., 1992). Receptor chimeras also aid in
mapping functional domains in receptors. α2
and β2 adrenoceptors are both activated by
epinephrine but differ in their G protein in-
teractions; the α2 adrenoceptor interacts with
Gi while the β2 adrenoceptor interacts with
Gs . Using differing chimeric constructs of α2
and β2 adrenoceptors, Kobilka et al. (1988)
showed that the receptor motif extending from
the N-terminus of transmembrane domain V
(TM-V) to the C-terminus of transmembrane
domain VI (TM-VI) was responsible for cou-
pling to Gs and activation of adenylyl cyclase.
Chimeric proteins combining GPCRs with G
protein α subunits have also been important for
investigating structural determinants of both
receptor and G protein activation and select-
ivity (Ward and Milligan, 2004). Chimeric
nicotinic-serotonergic receptors have been
prepared that show that the distinct subunits
from the 5-HT3 receptor and the nicotinic
α7 receptor, which are functionally distinct
LGICs, can couple together in a functionally Receptor
Binding
significant manner (Elsele et al., 1993). In this
1.1.13
Supplement 32
 case, the α7-V201-5HT3 chimera is sensitive
to nicotinic agonists, but not to serotonin.
The change in receptor function resulting
from point mutations is of more than scientific
interest, as a number of diseases have been
linked to receptor mutations (Coughlin, 1994).
Loss-of-function mutations can lead to re-
tinitis pigmentosa, color blindness, congenital
nephrogenic diabetes insipidus, and familial
hypocalciuric hypercalcemia, while constitu-
tively activating mutations have been associ-
ated with hyperfunctioning thyroid adenomas
and familial male precocious puberty.
ASSESSING RECEPTOR
FUNCTION
Radioligand Binding
The evaluation of NCEs for activity at re-
ceptors, uptake sites, or enzyme active sites
is done at a number of levels. These activities
may be predicted based on existing knowledge
of the diversity and function of the molecu-
lar target. Before 1970, efforts to character-
ize NCEs, and to understand disease etiology,
were directed at highly empirical tissue or an-
imal models that indirectly measured receptor
function as changes in the physical or bio-
chemical properties of a tissue or overt behav-
ior in an animal. That this pragmatic approach
was successful is evidenced by the contin-
ued stream of drugs that resulted. However,
since their precise mechanism of action was
unknown, it was nearly impossible to define
the factors responsible for efficacy of these
NCEs as distinct from those causing unwanted
side effects. The ratio between these two fac-
tors, the therapeutic index (TI), was therefore
poorly understood, making the search for com-
pounds free of major side effects difficult. The
development of radioligand binding assays in
the early 1970s by Cuatrecasas and Roth at
the NIH, Lefkowitz at Duke University, and
Snyder at Johns Hopkins University provided
a rapid means to evaluate compounds directly
for their ability to interact with a receptor
or enzyme, independent of efficacy (Williams
et al., 2005). As new assays were developed,
it became possible to assess the activity of a
compound against a battery of binding sites
to obtain an in vitro radioligand binding pro-
file for the compound. Binding assays are cur-
rently available for >100 different classes of
receptors and enzymes and are routinely used
to identify new receptor classes and subtypes.
Radioligand binding assays and the many
Receptors as receptor reporter systems they have spawned
Drug Targets
(UNITS 2.1 & 6.2) have provided biochemists
1.1.14
Supplement 32
with a rapid, cost-effective way of establish-
ing an SAR for a series of compounds. Be-
cause only minimal quantities of the NCE are
needed (1 to 5 mg), the information derived
from these assays is invaluable in the design
and synthesis of potent and selective agents.
The development of this screening technology
and its adaptation for high-throughput screen-
ing (HTS) has contributed significantly to the
drug discovery process and the development of
combinatorial chemistry. However, since bind-
ing assays measure only the recognition site on
the receptor, the intrinsic activity and efficacy
of the ligand are not revealed by this approach.
Functional Receptor Assays
The functional activity of a ligand is de-
termined by its ability to evoke a response.
Thus, an NCE may mimic the effects of an
endogenous agonist like acetylcholine (ACh)
or insulin by causing a muscle to contract or
blood sugar levels to fall. In such instances,
the degree to which the compound elicits a
response similar to that obtained from the nat-
ural ligand can reveal whether it is a full or
partial agonist. Conversely, a compound that
blocks the actions of either ACh or insulin
would be considered an antagonist. When us-
ing a tissue preparation to define such activity,
it is extremely important to delineate between
pharmacological and functional antagonism.
Pharmacological antagonism describes the
situation where a ligand antagonizes the ef-
fect of an agonist by blocking it directly at
its site of action. Functional antagonism can
yield data identical to those of a pharmaco-
logical antagonist, except that a functional an-
tagonist does not affect RL-complex forma-
tion but rather acts at a site distinct from the
receptor. An example is a hypothetical sys-
tem involving a GABAA receptor innervated
by a dopaminergic pathway that itself is un-
der the control of a cholinergic neuron (Fig.
1.1.4). From a pharmacological perspective,
any response ascribed to GABAA receptor ac-
tivation that is blocked by a compound could
be ascribed to GABAA antagonist activity.
However, a dopamine receptor antagonist or
nicotinic receptor antagonist would produce a
similar effect, thereby indirectly serving as a
functional antagonist of the GABAA site. Fail-
ure to appreciate this concept may lead to a
nicotinic antagonist being mistakenly charac-
terized as a GABAA receptor antagonist. This
example illustrates a major limitation of in
vivo compound evaluation in the absence of
in vitro data. Again, the hierarchical nature of
Current Protocols in Pharmacology
Figure 1.1.4 Pharmacological versus functional antagonism. (A) GABAA receptor activation produces a
signal (GABA release) which causes neuron B to produce a response. (B) Pharmacological antagonism:
blockade of the GABAA response at neuron B by a GABAA antagonist is a direct competitive effect. (C)
Functional antagonism: The same GABAA response on neuron B can be blocked in vivo by a nicotinic or
dopaminergic antagonist via interactions with upstream events in a pathway. In the absence of any further
data on the putative nicotinic or dopaminergic antagonist, they could be classified as GABAA antagonists.

compound evaluation, defining its selectivity
in vitro followed by the application of this in-
formation for interpretation of intact tissue and
in vivo studies, is the main reason for defining
the NCE site of action.
Functional activity may also be measured
biochemically by assessing ligand-induced
changes in receptor-linked second messenger
systems or gene-reporter constructs (UNIT 2.1).
This could include measurement of changes in
fluorescence of pH-, membrane potential- or
calcium-sensitive fluorophores (for LGICs or
GPCRs) or of melanophore-induced pigment
changes or luciferase-dependent light forma-
tion in systems linking these signals to second
messengers (for GPCRs). For enzymes, simi-
lar radiometric and spectrophotometric meth-
ods are used to assess either the catalytic dis-
appearance of substrate or the appearance of
product (UNIT 6.2).
Once an NCE is found to have functional
activity, it is important that the in vitro selec-
tivity be assessed in appropriate systems. For
instance, many different types of receptors are
known to alter cyclic AMP formation, as can
compounds that inhibit phosphodiesterase ac-
tivity. Thus, while a compound may be iden-
tified as selective at a particular receptor from
its binding profile, there is always the possibil-
ity it may alter second messenger production
by more than one mechanism. The use of ap-
propriate antagonists or agonists may help to
clarify this situation.
It is crucial to link the functional activity of
an NCE with the pathophysiology of the dis-
ease being targeted. Increased phosphatidyli-
nositol (Pl) turnover is a measure of muscarinic
cholinergic M1 receptor activation, a target that
has been associated with enhanced cognitive
performance in certain animal models. How-
ever, the hypothesis that M1 receptor agonists
enhance cognitive function must be proven in
appropriate animal models since it cannot be
assumed that the biochemical measure abso-
lutely predicts behavioral activity. Similarly,
while phosphodiesterase inhibitors alter car-
diac cyclic nucleotide metabolism, thereby in-
creasing contractile force, their use as positive
ionotropic agents for the treatment of conges- Receptor
tive heart failure must be assessed in an intact Binding

1.1.15
Current Protocols in Pharmacology Supplement 32
 animal model. In addition to validating the hy-
pothesis, evaluation of compounds in animal
models provides a measure of their bioavail-
ability.
The lack of activity of a compound in vivo
is more likely due to problems with bioavail-
ability and pharmacokinetics (PK; see UNIT
7.1) than to a mechanism of action unless the
former is ruled out. Frequently, compounds
for which no PK or bioavailability data are
available are administered to animals and as-
sessed in models where there is clear incon-
sistency between the activity being measured
and the half-life of the compound. Measuring
the anxiolytic potential of a peptide 15 min af-
ter administration is a flawed approach when
the half-life of the compound is measured in
seconds. The inability of an NCE to reach its
site of action due to lack of absorption, first-
pass metabolism, or lack of penetration across
the blood-brain barrier can limit its potential
as a drug candidate unless alternative dosage
forms can be used to overcome these obstacles.
While some of the factors affecting bioavail-
ability are understood (Navia and Chaturvedi,
1996), to date, there are no hard-and-fast rules
that can be applied across a chemical se-
ries. A similar caveat exists when addressing
toxicity.
Animal Models
There are several animal models that pro-
vide a reasonable approximation of what will
occur in humans when a compound enters clin-
ical trials. The spontaneously hypertensive rat
(SHR; UNIT 5.1) is a very useful model in pre-
dicting the hypotensive activity of a compound
in humans, provided the compound is bioavail-
able. Animal models of pain (e.g., mouse hot
plate; UNIT 5.7) and renal function (UNIT 5.21)
can predict activity in clinical trials. However,
in general, animal models of disease states
are generally retrospective, empirical tests for
measuring the effects of known active com-
pounds, rather than the disease state per se.
For example, the rat catalepsy model used to
assess potential antipsychotics is in fact an in
vivo model of dopamine receptor blockade.
Likewise, the induction of septic shock in mice
using lipopolysaccharide is only an approxi-
mation of the human condition and is open to
criticism since several compounds active in the
mouse model subsequently have been shown
to be ineffective in the clinic. This probably re-
lates to the complexity of the cytokine cascade
in humans.
Receptors as Transgenic animal models of human dis-
Drug Targets
ease states represent a potentially important
1.1.16
Supplement 32
approach in the effort to increase the relia-
bility of animal test procedures (Zambrowicz
and Sands, 2003). By introducing a foreign
gene into chromosomal DNA, a transgenic or
“knockout” animal can be produced in which
the expression of the native gene is inhibited.
The function of the targeted gene may then be
assessed in the living animal and may provide
a model of the human condition. In several
instances, the phenotype of the transgenic ani-
mal appears unchanged, indicating that the ex-
pression of the altered gene is not related to the
disease despite genomic evidence to the con-
trary. In others, because of the critical nature of
the targeted gene, the animals do not survive
when expression of the targeted gene is in-
hibited. Alternatively, since the transgenic ap-
proach requires that the altered gene be present
at birth, the animal may have sufficient func-
tional redundancy in the targeted system to
overcome any deficiency that results from the
transgene modification.
Newer approaches to creating animal ge-
netic models of human disease involve the
transfection of foreign genes into the germ
cell line that are subsequently only activated
in specific tissues (e.g., the tyrosine hydrox-
ylase Cre-loxP construct in striatum; Gelman
et al., 2003). Others are present in a quies-
cent state in the host genome but can be acti-
vated selectively once the animal has reached
adulthood to better approximate the onset of
the human disorder. Despite some limitations,
transgenic models can provide important in-
formation about the potential disease etiology.
For instance, overexpression of adenosine A1
receptors increases myocardial resistance to
ischemia, suggesting that this receptor may be
deficient in damaged heart muscle (Matherne
et al., 1997).
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Internet Resources
http://www.sigmaaldrich.com/Area of Interest/
Life Science/Cell Signaling/Sigma RBI
Handbook2.html
The Sigma-RBI Handbook of Receptor Classifica-
tion and Signal Transduction.
Contributed by Michael Williams and
Rita Raddatz
Worldwide Discovery Research
Cephalon, Inc.
West Chester, Pennsylvania
Current Protocols in Pharmacology