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

Muscle Fiber

Drugs only act here??What about drugsacting here, also??

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 SignalingIntercellular Signaling

Classes of cell-surface Classes of cell-surface receptorsreceptors

• Receptor must possessReceptor must possess structural and steric specificitystructural and steric specificity for a for a hormone and for its close analogs as well.hormone and for its close analogs as well.

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

• Hormone-receptor binding is Hormone-receptor binding is cell specificcell specific in accordance with target in accordance with target organ specificity.organ specificity.

• Receptor must possess aReceptor must possess a high affinity for the hormone at high affinity for the hormone at physiological concentrationsphysiological concentrations. .

•Once a hormone binds to the receptor, some recognizable Once a hormone binds to the receptor, some recognizable early early chemical eventchemical event must occur. must occur.

Criteria for hormone-mediated eventsCriteria for hormone-mediated events

• 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 = # 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

•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 Receptor occupancy, activation of target cell responses, kinetics of bindingresponses, kinetics of binding

•Activation of membrane receptors and Activation of membrane receptors and target cell responses is target cell responses is proportional to proportional to the degree of receptor occupancy.the degree of receptor occupancy.

•However, the hormone concentration at However, the hormone concentration at which which half of the receptorshalf of the receptors is occupied is occupied by a ligand (Kby a ligand (K

dd) is often lower than the ) is often lower than the

concentration required to elicit a concentration required to elicit a half-half-maximal biological responsemaximal biological response (ED (ED

5050))

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

Use the following numbers:

[L] = KD= 50% F.O.

[L] = 0.5 KD = 30% F.O.

[L] = 10x KD = 90%+ F.O.

[L] = 0= 0% F.O.

100

50

0 Ligand Concentration

Fra

ctio

nal

Occ

upan

cy

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 nMKD = 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.

0 5 10 20 25 30

100

50

0

Dose nM

A. Three drugs with presumably different B. Inverted U-shaped curve.receptor affinities and potencies.

-Same maximal effect.

Partial agonists1. 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.

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 sites).

A = agonist aloneB = 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• 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

Opioid receptor subtypesReceptor type µ-Receptor

1, 2, 3 ?? -Receptor

1, 2 ?? -Receptor1, 2 ??

Selective agonists endomorphin-1endomorphin-2DAMGO

[D-Ala2]-deltorphin I[D-Ala2]-deltorphin IIDPDPESNC 80DSLET

enadolineU-50488U-69593

Selective antagonists CTAP naltrindoleTIPP-ICI 174864

nor-binaltorphimine

Stopping the GPCR signal

• Endogenous GTPase within G 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 2-AR.– Activation of PKA/GRKs– Phosphorylation-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).

-arrestin binds with clathrin AP-2 binding site.– Complex internalizes into membrane-bound endosomes.– Endosomes internalizes– transient decrease in surface receptor number.

Bound

B/F

Receptor desensitization

-log [agonist] M0 10 9 8 7 6 5 4 3

% r

espo

nse

100

50

0

untreated

Chronic treatment

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.

Bound

B/F

Receptor down-regulation

-log [agonist] M0 10 9 8 7 6 5 4 3

% r

espo

nse

100

50

0

untreated

Chronic treatment

Receptor down-regulation