Receptor theory

• First postulated by John Langley (1878)

– Established after his experiments using nicotine and
curare analogues on muscle contraction.
• Isolated muscle fibers: pilocarpine (contraction) and atropine
(inhibition).
• Two compounds competing for a third, but unknown substrate.
• Furthered by Paul Ehrlich (1854-1915)
– Demonstrated that stereoselectivity was imperative in
drug-receptor signaling.
 John Langley
• In 1901, Langley challenged the dominant hypothesis that
drugs act at nerve endings by demonstrating that nicotine
acted at sympathetic ganglia even after the degeneration of
the severed preganglionic nerve endings.
• That year, Langley also discovered for himself a tool in the
form of renal extract (containing adrenaline) which
produced sympathomimetic responses when applied to
tissues exogenously.
• But it was not until 1905 that Langley published the results
of the decisive experiments using systemic injections of
curare and nicotine given to chicks. It was through these
experiments that Langley concluded the existence of a
receptive substance in striated muscle.
 Nerve/Muscle Endings

Drugs only act here??

What about drugs
acting here, also??

Muscle Fiber
 John Langley
• Langley concluded that a protoplasmic "receptive
substance" must exist which the two drugs
compete for directly. He further added that the
effect of combination of the receptive substance
with competing drugs was determined by their
comparative chemical affinities for the substance
and relative dose.
Intercellular Signaling
Classes of cell-surface
receptors
 Criteria for hormone-mediated events

• Receptor must possess structural and steric specificity for a

hormone and for its close analogs as well.

• Receptors are saturable and limited (i.e. there is a finite number of

binding sites).

• Hormone-receptor binding is cell specific in accordance with target

organ specificity.

• Receptor must possess a high affinity for the hormone at

physiological concentrations.

•Once a hormone binds to the receptor, some recognizable early

chemical event must occur.
• Affinity: The tenacity by which a drug binds to its receptor.
– Discussion: a very lipid soluble drug may have irreversible effects;
is this high-affinity or merely a non-specific effect?

• Intrinsic activity: Relative maximal effect of a drug in a particular

tissue preparation when compared to the natural, endogenous ligand.
– Full agonist – IA = 1 (*equal to the endogenous ligand)
– Antagonist – IA = 0
– Partial agonist – IA = 0~1 (*produces less than the maximal
response, but with maximal binding to receptors.)

• Intrinsic efficacy: a drugs ability to bind a receptor and elicit a

functional response
– A measure of the formation of a drug-receptor complex.

• Potency: ability of a drug to cause a measured functional change.

Receptors have two major properties: Recognition and Transduction
Recognition: The receptor protein must exist in a conformational state that allows for
recognition and binding of a compound and must satisfy the following criteria:

•Saturability – receptors exists in finite numbers.

•Reversibility – binding must occur non-covalently due to weak intermolecular forces (H-
bonding, van der Waal forces).

•Stereoselectivity – receptors should recognize only one of the naturally occurring optical
isomers (+ or -, d or l, or S or R).

•Agonist specificity – structurally related drugs should bind well, while physically
dissimilar compounds should bind poorly.

•Tissue specificity – binding should occur in tissues known to be sensitive to the

endogenous ligand. Binding should occur at physiologically relevant concentrations.
 The failure of a drug to satisfy
any of these conditions indicates
non-specific binding to proteins
or phospholipids in places like
blood or plasma membrane
components.
Receptors have two major properties: Recognition and Transduction

Transduction: The second property of a receptor is that the binding of

an agonist must be transduced into some kind of functional response
(biological or physiological).

Different receptor types are linked to effector systems either directly or

through simple or more-complex intermediate signal amplification
systems. Some examples are:

• Ligand-gated ion channels – nicotinic Ach receptors

• Single-transmembrane receptors – RTKs like insulin or EGF receptors
• 7-transmembrane GPCRs – opioid receptors
• Soluble steroid hormones – estrogen receptor
 Predicting whether a drug will cause a response in a
particular tissue

Factors involving the equilibrium of a drug at a

receptor.
• Limited diffusion
• Metabolism
• Entrapment in proteins, fat, or blood.
Response depends of what the receptor is connected to.
• Effector type
• Need for any allosteric co-factors – THB on tyrosine hydroxylase.
• Direct receptor modification – phosphorylation
 Receptor theory and receptor binding.

Must obey the Law of Mass Action and follow basic laws
of thermodynamics.
• Primary assumption – a single ligand is binding to a
homogeneous population of receptors

NH+3

COO-
 kon /k1

[ligand] + [receptor] [ligand • receptor]

koff /k2

• kon = # of binding events/time (Rate of association) = [ligand] •

[receptor] kon = M-1 min-1
• koff = # of dissociation events/time (Rate of dissociation) = [ligand •
receptor] koff = min-1
• Binding occurs when ligand and receptor collide with the proper
orientation and energy.
• Interaction is reversible.
• Rate of formation [L] + [R] or dissociation [LR] depends solely on
the number of receptors, the concentration of ligand, and the rate
constants kon and koff .
•At equilibrium, the rate of formation equals that of dissociation so that:

[L] • [R] kon = [LR] koff

KD = k2/k1 = [L][R]
[LR]
*this ratio is the equilibrium dissociation constant or KD.

KD is expressed in molar units (M/L) and expresses the affinity of a drug for a
particular receptor.
• KD is an inverse measure of receptor affinity.
• KD = [L] which produces 50% receptor occupancy
• Once bound, ligand and receptor remain bound for
a random time interval.
• The probability of dissociation is the same at any
point after association.
• Once dissociated, ligand and receptor should be
unchanged.
• If either is physically modified, the law of mass
action does not apply (receptor phosphorylation)
• Ligands should be recyclable.
 Receptor occupancy, activation of target cell
responses, kinetics of binding

•Activation of membrane receptors and

target cell responses is proportional to
the degree of receptor occupancy.

•However, the hormone concentration at

which half of the receptors is occupied
by a ligand (Kd) is often lower than the
concentration required to elicit a half-
maximal biological response (ED50 )
 Receptor Fractional Occupancy
F.O. = [LR]____ = [LR]___ *now substitute the KD equation.

[Total Receptor] [Rf] + [LR]

[R] = KD • [LR]  F.O. = [Ligand]

[L] [Ligand] + KD

100

Fractional Occupancy
Use the following numbers:
[L] = KD= 50% F.O.
50
[L] = 0.5 KD = 30% F.O.
[L] = 10x KD = 90%+ F.O.
[L] = 0= 0% F.O. 0
Ligand Concentration
Assumptions of the law of mass action.

• All receptors are equally accessible to

ligand.
• No partial binding occurs; receptors are
either free of ligand or bound with ligand.
• Ligand is nor altered by binding
• Binding is reversible
• Different affinity states?????
 Studies of receptor number and function
• We can directly measure the number (or density) of receptors in the LR complex.
• Ligand is radiolabeled (125I, 35S. or 3H). Selection of proper radioligand:
– Agonist vs. antagonist (sodium insensitive)
– Higher affinity for antagonists
– Longer to steady state binding
• Saturation binding curve-occurs at steady state conditions (equilibrium is
theoretical only).
• Demonstrates the importance of saturability for any selective ligand.
• Provides information on receptor density and ligand affinity and selectivity.
 Scatchard transformation
• Y-axis is Bound/Free (total radioligand-bound)
• X-axis Bound (pmol/mg protein)
• Straight lines are easier to interpret.
• The amount of drug bound at any time is solely
determined by:
– the number of receptors
– the concentration of ligand added
– the affinity of the drug for its receptor.
• Binding of drug to receptor is essentially the same as drug to
enzyme as defined by the Michelis-Menten equation.
However, not every ligand is
radiolabeled…What to
do?????
 Competition binding assays
• Allows one to determine a rough estimate of an unlabeled
ligand’s affinity for a receptor.
• Competitive or non-competitive.
• Introduction into the incubation mixture of a non-radioactive
drug (e.g. drug B) that also binds to R will result in less of R
being available for binding with D*, thus reducing the amount of
[D*R] that forms. This second drug essentially competes with D*
for occupation of R. Increasing concentrations of B result in
decreasing amounts of [D * R] being formed.
• Method:
– Single concentration of labeled ligand
– Multiple (log-scale) concentrations of the
unlabeled/competing ligand.
 Competition binding assays
• The concentration of inhibitor which displaces 50% of the
radiolabeled ligand is known as the IC50 for that drug.
• IC50 cannot be viewed as the “KD” of the inhibitor because it is
just an estimate.
• Ki = the equilibrium inhibitor dissociation constant.
– It is the concentration of the competing ligand that would
bind to 50% of sites in the absence of the radioligand.
• Ki can only be determined after the IC50 is known.
• Uses the equation of Cheng and Prusoff.

Ki = IC50
1 + [radiolabeled ligand]
Kd
Example: Find Ki of morphine in a preparation with 3H-diprenorphine.

IC50 = 100 nM Ki = 25 nM
[L] = 3 nM
KD = 1 nM
 Dose-response experiments.
• Measures the functional response of a drug, which is an indirect assessment of
receptor binding.
– Can be in vitro, in vivo, or ex vivo.
• Is response directly proportional to receptor occupancy????
– Clarke’s Theory: the effect of a drug is proportional to the fraction of
receptors occupied by the drug and maximal response occurs when all
receptor are bound. Is this true????
• Actually, more is not necessarily better.
 Fractional response
• Equation for fraction response for Drug A:
• Rf is the fractional response for any concentration of agonist.
• The dose producing the maximum effect (Emax ) is termed the maximum effective
dose, whereas the concentration of agonist producing the half-maximal response is
termed the EC50 .
• If the agonist concentration is expressed in log terms then the resultant dose-
response curve is sigmoid shaped.
• A concentration of agonist 10 fold higher than its EC50 would produce a response
that is 90% of Emax whereas a concentration of agonist 100 fold higher than its EC50
would produce a response 99% of Emax .
 However, not all agonists acting at the same
receptor produce the same maximal response.

100

50

0 5 10 20 25 30
Dose nM
A. Three drugs with presumably different B. Inverted U-shaped curve.
receptor affinities and potencies.
-Same maximal effect.
 Partial agonists
1. Some agonists never elicit a maximal
response (compared to the
endogenous agonist) even when
nearly all of the receptors are
occupied.
However, the EC50 for these are
remarkably close to full agonists:
1. Similar potency, but lower efficacy:
Intrinsic activity = 0~1
2. High efficacy drug: need to occupy
fewer receptors to produce a response
than one with lower efficacy.
3. Why????
several conformation changes
can occur by different agonists.
1. Similarly, partial agonists will elicit
a very low or no measurable
functional response even when a
significant number of receptor are
occupied.
Partial agonists can act as functional antagonists
when in competition with higher efficacy agonists.

• Methadone for heroin abuse treatment.

– Used to “wean off” abused drugs.
• Basically competition between the full and partial agonist.
 Receptor antagonists.

• Prevent agonist-mediated responses by preventing a

drug from binding and eliciting its normal response.
• Intrinsic activity = 0.
• No sensitivity to Na+ or GTP.
• Antagonists are measured by the selectivity, affinity for
their receptor, and potency.
 Receptor antagonists
Competitive antagonist.
• Reversible or irreversible.
• Bind to the same site as the
endogenous ligand or agonist.
• Can be over come!
• Their presence produces a right-
ward shift in both the binding
and dose-response curves.
• No change in Emax .
• Similar dose-response curve
shapes indicates the presence of
a competitive agonist
(competing for the same binding A = agonist alone
sites). B = antagonist (one concentration)
A+B = agonist + antagonist
 Non-competitive antagonist
• Does not prevent formation of the
DR complex, but impairs the
conformation change which
triggers a response.
• Bind to a site different than the
agonist binding site at an allosteric
site (use a hemoglobin example.).
• Cannot be overcome by adding
more agonist
• Emax and Bmax are reduced but EC50
remains the same for the unaffected
receptors.
• Dose-response curves will have
different shapes indicating
different binding sites.
 Irreversible antagonists.
• Binds in an irreversible manner, usually by covalent
modification of the receptor.
• EEDQ (non-selective)
• N-ethylmalemide (NEM) or other sulfhydryl or
alkylating agents (non-selective).
• Antibodies
• Molecular control (mutation) – EXAMPLE
• Prevents binding at the atomic level.
• Effectively and practically lowers the number of
receptors capable of binding an agonist.
• Adding more agonist is useless
• Only cure: Make New Receptors by Protein
Synthesis.
 Receptor subtypes
100
• First learned for the histamine
receptor.
contraction
– histamine activation by agonist % response
produces smooth muscle
contraction. 50 Gastric secretion

• The residual activity in gastric

secretion, even in the absence of
muscle contraction, indicated the
Contraction + antagonist
presence of histamine-sensitive 0
receptors. .001 .01 1 10 100 1000
• Conclusion = Receptor Subtypes. Log [histamine]
– Receptor subtypes are
characterized by:
– Binding differences (selective
ligands)
– Function
– Molecular cloning analysis
revealing amino acid
differences.
 Opioid receptor subtypes
Receptor type µ-Receptor δ -Receptor κ -Receptor
µ 1, µ 2, µ 3 ?? δ 1, δ 2 ?? κ 1, κ 2 ??
Selective agonists endomorphin-1 [D-Ala2]-deltorphin I enadoline
endomorphin-2 [D-Ala2]-deltorphin II U-50488
DAMGO DPDPE U-69593
SNC 80
DSLET
Selective antagonists CTAP naltrindole nor-binaltorphimine
TIPP-ψ
ICI 174864
 Stopping the GPCR signal
• Endogenous GTPase within Gα subunit
• Proteolysis of receptor-rare
• NT re-uptake or enzymolysis
• RGS proteins-regulators of GTPase
• Receptor internalization/down-regulation.
 Receptor desensitization
• A loss of agonist affinity, but not receptor number after
chronic agonist stimulation.
– Best example is β 2-AR.
– Activation of PKA/GRKs
– Phosphorylation
− β -arrestin
• uncoupling of receptor and G-protein
• results in a rightward shift of the binding curve:
DESENSITIZATION.
• KD of isoproterenol (1  100 nM) goes up
– affinity goes down
– number of receptors does not change (Bmax does not change).
 β -arrestin binds with clathrin AP-2 binding site.
– Complex internalizes into membrane-bound endosomes.
– Endosomes internalizes
– transient decrease in surface receptor number.
 Receptor desensitization

B/F

Bound
 100 untreated
% response

50 Chronic treatment

0
0 10 9 8 7 6 5 4 3
-log [agonist] M

Receptor desensitization
Adapted from Lefkowitz, 1998 (JBC, vol., 273)
 Receptor down-regulation
• Proteolytic degradation of receptor
– producing a net loss in total cell receptor number.
• PKC involvement during endocytosis
• Bmax can decreases (~60%); KD remains the same
• Use of endosomes and lysosomes.
 Receptor down-regulation

B/F

Bound
 100 untreated
% response

50 Chronic treatment

0
0 10 9 8 7 6 5 4 3
-log [agonist] M

Receptor down-regulation