Lecture on Receptors

1. Shri Sharda Bhavan Education Society’s
Nanded Pharmacy College
Lecture on,
RECEPTORS
By
Dr. Shriniwas K. Sarje
Asso. Professor & HOD Department of Pharmacology

2. 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.

3. 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.

4. Nerve/Muscle Endings
Muscle Fiber
Drugs only act here??
What about drugs
acting here, also??

5. 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.

6. Intercellular Signaling

7. Classes of cell-surface receptors
guanosine nucleotide-binding-protein

8. • 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.
Criteria for hormone-mediated events

9. • 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.

10. 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 concs.

11. 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 – Receptor tyrosine kinase (RTKs) like
or insulin epidermal Growth Factor (EGF) receptors
• 7-transmembrane GPCRs – opioid receptors
• Soluble steroid hormones – estrogen receptor

12. 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
• Direct receptor modification – phosphorylation

13. 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-

14. • 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.
kon/k1
[ligand] + [receptor] [ligand  receptor]
koff/k2

15. •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 = which produces 50% receptor occupancy

17. • 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.

18. 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)

19. 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?????

20. • 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.

21. 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.

22. Dose-response experiments.
• Measures the functional response of a drug, which is an indirect
assessment of receptor binding.
– Can be in vitro, in 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.

23. 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.

24. However, not all agonists acting at the same
receptor produce the same maximal response.
Three drugs with presumably different
receptor affinities and potencies.
-Same maximal effect.

25. 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:
2. Similar potency, but lower efficacy:
Intrinsic activity = 0~1
3. High efficacy drug: need to occupy
fewer receptors to produce a response
than one with lower efficacy.
4. Why????
several conformation changes
can occur by different agonists.
5. Similarly, partial agonists will elicit
a very low or no measurable
functional response even when a
significant number of receptor are
occupied.

26. 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.

27. 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.

28. Receptor antagonists
Competitive antagonist.
• Reversible
• 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 sites).
A = agonist alone
B = antagonist (one concentration)
A+B = agonist + antagonist

29. 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
• Cannot be overcome by adding
more agonist
• Emax reduced but EC50 remains the
same for the unaffected receptors.
• Dose-response curves will have
different shapes indicating
different binding sites.

30. Irreversible antagonists.
• Binds in an irreversible manner, usually by
covalent modification of the receptor.
• sulfhydryl or alkylating agents (non-selective).
• Antibodies
• 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.

31. Receptor subtypes
• First learned for the histamine
receptor.
– histamine activation by
agonist produces smooth
muscle contraction.
• The residual activity in gastric
secretion, even in the absence of
muscle contraction, indicated the
presence of histamine-sensitive
receptors.
• Conclusion = Receptor Subtypes.
– Receptor subtypes are
characterized by:
– Binding differences (selective
ligands)
– Function
– Molecular cloning analysis
revealing amino acid
differences.
% response
100
50
0
Log [histamine]
.001 .01 1 10 100 1000
contraction
Gastric secretion
Contraction + antagonist

32. Opioid receptor subtypes
Receptor type µ-Receptor
m1, m2, m3 ??
d -Receptor
d1, d2 ??
k -Receptor
k1, k2 ??
Selective agonists endomorphin-1
endomorphin-2
[D-Ala2]-deltorphin I
[D-Ala2]-deltorphin II
Enadoline
Selective antagonists Naloxone Naltrindole nor-binaltorphimine

33. Receptor desensitization
• A loss of agonist affinity, but not receptor number after
chronic agonist stimulation.
– Best example is b2-AR.
– Activation of PKA
– Phosphorylation
• uncoupling of receptor and G-protein
• results in a rightward shift of the binding curve:

34. Receptor down-regulation
• Proteolytic degradation of receptor
– producing a net loss in total cell receptor number.
• PKC involvement during endocytosis