Receptors: radioligand binding and data analysis

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Receptors radioligand binding and data
analysis September 9 (900 1000 am), 2005
Vsevolod V. Gurevich, Ph.D. (my short name is
Seva) vsevolod.gurevich_at_vanderbilt.edu
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• Signal Transduction
• definition
• 1st step receptors ? bifunctional molecules
• receive information
• do something with that information

• In this section, will focus on cell surface
  receptors that mediate the response to
  extracellular signals
• from the environment (light, odorants, etc.)
• paracrine (secreted agent is local mediator)
• synaptic (basis for neurotransmission)
• endocrine (blood borne to distant target cell)

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Receptors come in many flavors different
structural motifs functional styles
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Agents acting at receptors can elicit different
biological effects
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Effect, basal response
100
50
agent

• receptors provide specificity
• example a vs. b adrenergic receptors
  (described by Ahlquist)
• binding precedes action
• reversible biological responses predict
  reversible binding

• biological response is a function of
• affinity (related to KD)
• available concentration of agonist
• efficiency of coupling/amplification

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The interaction of an agonist with a receptor can
be depicted
response ?agonistreceptorKAe where KA
equilibrium affinity constant (discussed in more
detail later) and e efficiency of
receptor-effector coupling leading to biological
response Disease is manifest by change in
response.
Classic endocrine disorders due to Dagonist
availability DKA consequence of receptor
mutations, post-translational modifications
(especially phosphorylation) De typically due
to post-translational modifications mediated by
downstream kinases, phosphatases DR mutation
perturbation of expression or turnover by other
biological processes
Numerous biological processes reflect changes in
signal transduction with development, disease,
aging, etc., it has been necessary to obtain
quantitative data on RECEPTOR OCCUPANCY and
SIGNALING EFFICIENCY
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Quantitative Descriptors of Ligand-Receptor
Interactions (ligand could be hormone,
neurotransmitter, growth factor,synthetic drug,
etc., acting as agonist, partial agonist,
antagonist, or inverse agonist. The same analysis
and math is applicable to any small molecule
binding to any protein, and even to
protein-protein interactions)
Note the higher the KD value, the lower the
affinity, and vice versa. KD related to
OCCUPANCY EC50 related to RESPONSE! Because of
amplification of signal transduction pathways KD
typically gtgt EC50
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• How do you know binding detected is due to a
  biologically relevant receptor?
• CRITERIAbased on functional properties of
  receptor
• saturability
• specificity characteristic of the biological
  response
• kinetics (on/off of binding) consistent with
  rate of initiation termination of biological
  response)

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• It can be calculated that DR/D separation must
  be completed within 0.15t1/2 in order to avoid
  losing more than 10 of the DR complex. The
  relationship between KD and separation time is
  therefore calculated
• For a typical centrifugation/vacuum filtration
  experiment the practical lower limit of affinity
  (upper limit KD) is 10 nM (10-8 M).

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Saturability Number of receptors per cell or
membrane is finite. Assessed by saturation of
binding sites. Binding should reach maximum with
increasing concentrations of ligand.
At equilibrium, rate of association rate of
dissociation i.e. konLR koffLR KD
koff/kon LR/LR Rtotal R
LR Therefore LR RtotalL/(KD L)
This equation describes rectangular hyperbola
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Since maximum binding (Rtotal or Bmax) is never
achieved, i.e. curve is asymptotic, computer
analysis (non-linear regression) is used to
compute Bmax. Alternatively, linear
transformation of the data is used (rely on
SLOPE, INTERCEPTS, to get quantitative values of
interest).
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Explanation of math involved (dont get scared,
it is very simple) Mass action law KD
RL/RL Rtotal Bmax R RL gt R
Bmax - RL Bound B RL Free F L
(assuming that L gtgt RL) Thus, mass action
low can be expressed as KD (Bmax
-B)(F)/B Hence (KD)B (Bmax -B)F Rearrange as
B/F (Bmax -B)/ KD, or B/F -1/ KD (B- Bmax)
This is Scatchard equation plotted on the
previous slide
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What do you do if your Scatchard does not look
like a straight line?

• Make sure that your data are reliable and the
  problem of non-linear Scatchard is real.
• Use other methods to discriminate between
  negative cooperativity and multiple independent
  sites/different affinity states (discussed later).

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• Computer-assisted analysis ? resolving complex
  binding into individual KD values, Bmax values,
  etc.
• mathematical algorithm must fit biology
• (e.g. most ligand binding algorithms assume AT
  EQUILIBRIUM)
• need independent experimental strategies to
  demonstrate existence of
• receptor affinity states
• receptor subtypes
• allosteric modulation of receptor binding (e.g.,
  G protein-coupled receptor interactions with G
  proteins or arrestins)

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Specificity Assessed by competition binding
studies Example b-adrenergic receptor Should
bind epinephrine and structural analogs should
exhibit a potency order in binding these
Can calculate KD from these data EC50 for
isoproterenol is 10-8M. But this is not its KD
because it is competing for 125I
propranolol. In competitive binding there are two
equilibria
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Complexity in Ligand Binding G protein-modulated
receptor affinity states as an example
Complexity in ligand binding is manifest by

• shallow or steep competition binding curves

• simple
• from 10-90 competition over an 81 fold
  concentration range of competitor
• manifestation of one ligand interacting with a
  single receptor with a single and constant
  affinity in a reaction that has reached
  equilibrium

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Complexity in Ligand Binding G protein-modulated
receptor affinity states as an example

• G protein-regulated systems have cycles of
• protein-protein association/dissociation cycles
• GTP hydrolysis cycle
• G protein subunit association/dissociation cycle

Partial reaction
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Complexity in Ligand Binding G protein-modulated
receptor affinity states as an example
Example epinephrine competing for radiolabeled
antagonist binding
agonist GTP or GTP analog
agonist alone

• GraphPad Prism and other computer software permit
  quantitation of
• RHi - higher affinity state
• RLo - lower affinity state
• KDHi, KDLo ? receptor affinity for particular
  agonist at RHi,RLo

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Kinetic Binding Analysis Dissociation Kinetics

• Dissociation rates can be measured in two ways
  1) by infinite dilution, the reaction mixture is
  diluted to an extent where no further association
  can take place, or by excess cold ligand where
  further association is blocked by excess cold
  competitor.
• These two methods will give different results if
  there are complex binding phenomena.

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• Assumptions
• Reaction is reversible
• 1 receptor
• 1 ligand
• 1 affinity
• What are the possible reasons for nonlinear
  dissociation
• Multiple receptor subtypes
• Multiple affinity states
• cooperativity (negative, if the plot has the same
  form as that observed for multiple affinity
  states/subtypes).
• The method of dissociation can distinguish
  between cooperativity and the other
  possibilities. At infinite dilution D is low.
  For excess cold ligand D will be a constant
  high value, so that only the low affinity site
  will be observed for negative cooperativity (high
  for positive cooperativity). In the case of
  multiple receptor subtypes of affinity states the
  result will be unchanged no matter which method
  is used.
• Thus this method allow a differential diagnosis
  for cooperativity versus multiple
  receptors/affinity states.

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• What properties would you expect of distinct R
  subtypes?
• non-interchangeable
• might vary in relative density to one another at
  various states of development, from various
  tissues

How do you readily distinguish between two
independent receptor populations versus
allosteric regulation/receptor affinity
states? - can exploit dissociation kinetics
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Kinetic Binding Analysis Association Kinetics

• The third criterion expected for the binding of a
  radiolabeled drug or hormone(D) to the
  physiologically relevant receptor is that the
  time course of binding should correspond to or
  precede the time course characteristic of the
  physiologic effect elicited by D.
• The rate of formation of DR can be described as
• The rate of the forward reaction is affected by
  both D and R.

D R
D R
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Reading

• Cell Surface Receptors A Short Course in Theory
  and Methods, Lee Limbird
• history in chapter 1
• selections from chapters 3 4

Ligand-receptor complexes origin and development
of the concept. Irving M. Klotz. J. Biol. Chem.
279, 1-12 (2004).
You may also find the following web site very
helpful http//www.unmc.edu/Pharmacology/receptor
tutorial/