IGP Cell Signaling

1
IGP Cell Signaling January 21, 2004, 900 1000
am Vsevolod V. Gurevich, Ph.D. (my short name is
Seva) vsevolod.gurevich_at_vanderbilt.edu (based on
materials developed by Lee Limbird)
2

• Signal Transduction
• definition
• 1st step receptors ? bifunctional
• receive information
• do something with that information

• In this section, will focus on signal
  transduction mediated by cell surface receptors
• 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)

3
Receptor Concept
Receptive substanceJ. N. Langley (1852-1926)

• Receptor Concept- Paul Ehrlich (1854-1915)
• distribution of lead in the body
• preferential distribution in the brain
• differential staining of tissues by dyes
• vital staining (acidophilic/basophilic)
• side chain theory for basis of immune
• response
• arsenicals to treat trypanosomes
• - relying on differential metabolic rates of
  host/invader (origin
  of chemotherapy)

4
Receptor Concept

• J. N. Langley
• mutual antagonism depends on relative quantities
  of drugs added, with limits
• drugs can form complexes according to some
  law, based on
• relative concentration
• relative affinity

5
Receptor Concept
Not everyone bought into the receptor concept

• H. H. Dale (1875-1968)
• Believed differential effectiveness of adrenaline
  analogs in mimicking sympathetic functions could
  be due to a CHEMICAL PROCESS
• did not necessarily imply existence of receptors
  on target tissues
• Favored distributive vs. interactive properties
  of a drug in determining target cell selectivity

6
Adrenergic Receptor Diversity

• Raymond Ahlquist (1948)
• Two adrenergic receptor populations
• a smooth muscle contraction
• norepi gt epi gt isoproterenol
• b smooth muscle relaxation
• cardiac muscle contraction
• isoproterenol gt epi gt norepi

Key Finding differential affinity! (hard
to reconcile as solely a distributive property)

• Later
• a blocked by phentolamine
• b blocked by propranolol

7
Receptors come in many flavors different
structural motifs functional styles
8
Agents acting as receptors can elicit different
consequences on a biological effects
200
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

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

9
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
10
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)
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
11

• How do you know binding detected is due to a
  biologically relevant receptor?
• CRITERIAbased on functional properties of
  receptor
• saturable
• specificity characteristic of the biological
  response
• kinetics (on/off of binding) consistent with
  rate of initiation termination of biological
  response)

12
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
13
Saturability Number of receptors per cell or
membrane is finite. Assessed by saturation
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
14
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).
15
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
16

• 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 (will return to later)
• receptor subtypes
• allosteric modulation of receptor binding (e.g.,
  G proetin-coupled receptor interactions with G
  proteins or arrestins)

17
G Protein-Coupled Receptors and the Concept of
Second Messengers
This part of our discussion will be from an
historical perspective, to give you insights into
the thinking processes that underlie discovery.
We also need to introduce GTP-binding proteins as
transducers of signaling to return to the concept
of affinity states.
Understanding the effects of epinephrine and
glucagon on the liver
Adding epinephrine (adrenaline) or glucagon to
liver preparations ? glycogenolysis (glycogen
breakdown) ? glucose production
epi ? animal ? increased glucose production
(increased glucose in circulation)
epi ? liver ? increased glucose production
epi ? liver ? increased glucose production
epi ? hepatocytes ? increased glucose production
(lowered glycogen)
Earl Sutherland identified a substance, cyclic
AMP (cAMP), that was elevated in extracts of
liver treated with epinephrine. Propranolol (a
b-adrenergic antagonist) blocked cAMP production.
Hypothesis cyclic AMP mediates effects of
epinephrine on glycogenolysis in the liver.
18
Criteria to test hypothesis that cAMP SECOND
MESSENGER of epinephrine (and glucagon) action
(distinguishing concomitant from causal)

• If cAMP indeed mediates action of epinphrine,
  then agents which prevent cAMP breakdown will
  enhance epinephrine action (e.g. theophylline
  caffeine other phosphodiesterase inhibitors).

• If cAMP indeed mediates action of epinphrine,
  then agents which prevent cAMP breakdown will
  enhance epinephrine action (e.g. theophylline
  caffeine other phosphodiesterase inhibitors).

• If cAMP indeed mediates action of epinphrine,
  then agents which prevent cAMP breakdown will
  enhance epinephrine action (e.g. theophylline
  caffeine other phosphodiesterase inhibitors).
• Analogs of cAMP that can permeate the cell
  membrane will mimic the actions of epinephrine in
  its absence, i.e. dibutyryl cyclic AMP (db-cAMP).

• If cAMP indeed mediates action of epinphrine,
  then agents which prevent cAMP breakdown will
  enhance epinephrine action (e.g. theophylline
  caffeine other phosphodiesterase inhibitors).
• Analogs of cAMP that can permeate the cell
  membrane will mimic the actions of epinephrine in
  its absence, i.e. dibutyryl cyclic AMP (db-cAMP).
• Epi should increase production of cAMP in broken
  cell preparations

Purification of hepatocyte membranes eliminates
effects of epi on cAMP production.
cAMP SECOND MESSENGER concept won Sutherland
the Nobel Prize
19
What could be the explanation for the loss of
cAMP production in response to epinephrine
following purification of hepatocyte membranes?

• technical explanation(s)
• - loss of catalytic activity for ATP ? cAMP
  during membrane preparation

• biological explanation(s)
• some factor/cofactor(s) lost upon membrane
  purification
• add back cytosolrestore response! cytosolic
  factor dialyzable (low molecular weight)
  resistant to protease treatment
• - factor that restored activity GTP, 10-7 M (ATP
  as substrate 1mM)

findings of Rodbell and colleagues (Martin
Rodbell shared Nobel Prize with Al Gilman for the
identification of GTP-binding proteinsG proteins)
20
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

21
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
22
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

23
Complexity in Ligand Binding G protein-modulated
receptor affinity states as an example
What would be your expectation for competition
binding studies of antagonists? inver
se agonists? Evaluated in the absence and
presence of added GTP/ GTP analogs re
positions of the curves shapes of
the curves
24
How does GTP work? Evolution of the models
25

• 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
26
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/