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What are the criteria for a
receptor?
• Capacity to bind a ligand (not unique)
• Membrane associated; induce biological
response when bound (not unique)
• Do not change the ligand by binding
(not unique)
• True neurotransmitter receptors do all
the above and mediate the response to
the transmitter.
Radioligand binding assay
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Tissue homogenate
High activity radioactive labeled ligand
Unlabeled ligand
Total binding measured in the presence of
increasing concentrations of labeled ligand
• Non-specific binding measured as above but
in the presence of excess unlabeled (cold)
ligand
• Specific binding is the subtraction of the two
Criteria defined by radioactive binding
assays: A Specific Receptor
• Saturability
– Very small amount of receptor in tissue
• Specific and high affinity
– Found in biologically relevant sites
– High receptor occupancy with low, physiologic
concentrations of ligand
• Biologic relevance
– Distribution of receptor matches distribution of ligand
– Binding or displacement of drugs should correlate with
the potency of those drugs
• Reversibility
– Timing and recovery of ligand unchanged
Binding characteristics
• Bmax: density of receptors
• kD: 1/affinity of receptor for the ligand
• Binding according to law of mass action
[L] + [R]
[LR]
Rate of reaction proportional to [ ] reactants
K1=[L]*[R]
K2=[LR]
At equilibrium, k1=k2.
Substituting, k1/k2=[L]*[R]/[LR]=kD, the equilibrium
dissociation constant
Scatchard Transformation
Displacement or indirect binding
Hill Plot
Hill Plot-Cooperativity
Some issues with radioligand
binding data
• Maximal response can be achieved with only
a portion of receptors bound
– Spare receptors
• Agents that bind equally may yield different
biological response
– Full agonist, partial agonist, antagonist
– Efficacy or intrinsic activity
• Desensitization: decreased response with
continuous exposure to drug (binding
unchanged)
Two state model
• Receptors go between active and
inactive state
– Agonists prefer active state
– Efficacy (response) determined by state
preference (ratio of affinity for two states)
– Antagonists prefer inactive state (no
efficacy)