isolation of receptors

7/28/2019 Isolation of Receptors

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by Lee Eun Jin

RECEPTOR ISOLATION,IDENTIFICATION AND

CHARACTERISATION (Methods)


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affinity columns with biotinylated ligands more systematic proteomics analysis of GPCR-associated protein complexes

was conducted using receptor-specific antibodies (7). However, the general application of these approaches was limited by the availability of adequate tools (labeled ligands, antibodies, etc.) for each GPCR. Later on, isolated intracellular domains were widely used to identify GPCR-associated proteins either as bait in yeast two-hybrid screens or to generate affinity matrices for the purification of interacting proteins from cell extracts (8 11). Using the entire C-tail of the 5HT2c receptor expressed as GST fusion protein, more than 15 proteins have been identified (12). Furthermore isolated protein-protein interaction motifs such as PDZ domain recognition motifs of GPCRs have been successfully used to identify interacting partners of the PDZ domain recognition motifs of the 5HT2a, 5HT2c, and 5HT4 receptors (13, 14).


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Radioligand

A radioligand is a radioactivelylabeled drug that can associate witha receptor, transporter, enzyme, or

any site of interest.

Measuring the rate and extent ofbinding provides information on thenumber of binding sites, and theiraffinity and accessibility for various

drugs


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Radioligand binding can beused to

1. Characterize receptors

2. Study receptor dynamics and localization;

3. Identify novel chemical structures that

interact with receptors4. Define ligand activity and selectivity in

normal and diseased tissues.


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Contd..

This simple model assumes; All receptors are equally accessible to ligands.

Receptors are either free or bound to ligand. Itdoesnt allow for more than one affinity state, or

state for partial binding. Binding dose not alter the ligand or receptor.

Binding is reversible.


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Factors to consider indesigning receptor binding

experiments1. Identify an appropriate radioactiveligand to use for the experiment.

2. Tissue preparation to be used inthe experiment

3. Identify a method for separatingbound from free.

4. Identify a method for distinguishingspecific from non-specific binding.


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Criteria for selecting aradioactive ligand

a. The specific activity of the radioligandshould be high enough to detect thereceptor in the tissue being studied.

b. The radioligand should have a high affinityfor the receptor.

c. The radioligand should have a high degreeof selectivity for the receptor being studied.

d. The radioligand should be chemically stablein the assay media during the bindingreaction.

e. The radioligand should be pure


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Radioligand binding couldbe

1. SpecificThe site that

we want to study is referred to as

the SPECIFIC SITE.

2. Non-specific

All other sites are called NON-SPECIFIC SITE.


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SATURABLE and NON- SATURABLEbinding

SATURABLE binding sites are always present in

finite amounts. Another way of thinking of this isthat if you add enough ligand all of the sites willbe occupied with ligand.

Examples of saturable sites;

Specific binding sites are always saturable All Receptors

NON SATURABLE binding sites are sites that arepresent in essentially infinite amounts. No matter

how much ligand you add, not all of the sites willbe occupied with ligand. The site is non-saturable.

Examples of non- saturable sites;

Low affinity tissue binding sites

Binding to test tubes and glass fiber filters


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Theoretical Basis for Characterizing Receptorsusing Saturation Radioligand Assays

Radioreceptor assays were first developedin the early 1970s. They were based on twovery simple, but very elegant concepts.

If a ligand had high affinity for a

macromolecular target (as had been shown byclassical pharmacological studies over manydecades), it should be thermodynamicallypossible to measure the binding of the

ligand to the receptor without the needto perform equilibrium dialysis as long asone could separate the ligand-receptorcomplex from the free ligand.

By labeling ligands with appropriateradioactive atoms, one could detect the li and-


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Major Types of RadioligandStudy

1. Saturation binding experiments : measureequilibrium binding of various concentrations ofthe radioligand. Analyze the relationship betweenbinding and ligand concentration to determine thenumber of sites Bmax, and the ligand affinity, Kd.

2. Competitive binding experiments: measureequilibrium binding of a concentration ofradioligand at various concentrations of anunlabeled competitor. Analyze these data to learnthe affinity of the receptor for the competitor.

3. Kinetics experiments: measure binding atvarious times to determine the rate constants for


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Saturation binding experiments

In a saturation experiment increasing concentrationsof a radioactive ligand are allowed to bind to thesteady-state conditions occur.

After reaching steady state, the bound ligand isseparated from the free ligand. The most widelyused methods for separation of bound ligand fromfree ligand are filtration and centrifugation.

The amount of ligand bound to the filter or trappedin the pellet is measured. A radioactive ligand isused because the radioactivity of low concentrationsof ligand can be detected in filters or membranepellets.


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Purpose of the saturation experiment

1. To determine the affinity or Kd of a radioligandfor receptor.

Kd is the equilibrium dissociation ofradioactive ligand required to occupy 50% ofthe receptors.

1. The density (Bmax) of a specific receptor orreceptor subtype in a given tissue.

Bmax is the total number of receptor sites inthe tissue being studied. It occurs whenthe all receptor molecules are occupies byradioactive drug.


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Methods used to determine Kd andBmax from a saturation experiment

1. Saturation curve

2. Rosenthal plot (commonly referredto as a Scatchard plot)


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1. Saturation curve

These experiments are called saturationexperiments because at the higherradioligand concentrations all of thereceptor molecules are occupied

(saturated) by radioactive ligand.

Results of the saturation experiment can

be plotted with BOUND (the amount ofradioactive ligand that is bound to thereceptor) on the Y axis and FREE (the freeconcentration of radioactive ligand) on the

X axis.


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Contd

The resulting graph is ahyperbola and is called asaturation carve.

Bmax is the maximalbinding which isapproached asymptoticallyas radioligandconcentration is increased.

Bmax is the density of thetissue being studied.

Kd is the concentration of

ligand required to occupy50% of the bindin sites.


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Contd

By fitting the data to the equation fora saturation curve.

Getting an accurate estimate of Kd

and Bmax from this graph by eye isdifficult.

The curve is usually analyzed by

nonlinear regression analysis.

B=Bmax* FKd +F

where B is Boundand F is Free


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2. Rosenthal plot

The data from thesaturation experimentcan be plotted withBound/Free on Y axis

and Bound on the X axis. Single site binding data

can be analyzed bylinear regression to give

straight line. The slope of line is -1/Kd

and the X-intercept isBmax. This is a

Rosenthal plot.


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Contd

Advantages of Rosenthal plot It is easy to visually compare two sets of

data on a Rosenthal plot.

If the Kd for both sets of data are similarthe slopes will be similar.

If the Bmax changes then the X interceptwill change.

Two-site fit to the data can be visualizedmore easily with a Rosenthal plot thanwith a saturation curve.


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2) Competition experiments

Many a times, ligand for receptors arenot available in a radioactive form.Since they are unlabeled there is no

way to directly measure their affinityfor the receptor.

The affinity of the unlabeled ligand forthe receptor can be determinedindirectly by measuring its ability tocompete with a radioactive ligand for

the receptor.


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Contd

In a competition experiment variousconcentrations of an unlabeled ligandare allowed to compete with a fixed

concentration of a radio labeledligand for a receptor.

As the concentration of unlabeled

ligand increases, the amount ofradioligand bound to the receptordecreases.


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Contd

The competitive inhibitorcan be either an agonistor an antagonist.

It is called a competitive

inhibitor because itsvalue is determined bymeasuring the ability ofthe unlabeled drug tocompete with a radiolabeled drug for thereceptor.

The Ki for an unlabeleddrug should be the same

as the Kd value obtained


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Purpose of competition experiments

Competition experiments can beused to determine the affinity of theunlabeled ligand for the receptor.

The affinity of an agonist for areceptor.

The pharmacological characteristicsof subtypes of a particular receptor.

The classification of receptorsubtypes in a tissue.


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Application of Radioligand bindingassay

Methods used to identify thepharmacological profile for a specificsubtype of a receptor

To initial identify the pharmacological

profile of a receptor subtype it is importantto first have a tissue which selectivelyexpresses only that receptor subtype.

Competition studies are done to determinethe affinity of a large number of drugs for

the receptor. It is important to use drugs which have both

a high and low affinity for the receptor. The affinity of these drugs for the receptor

define the pharmacological profile for therece tor.


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Methods used to identify the receptorsubtypes in an unknown tissue First, the pharmacological profiles for the

known receptor subtypes need to bedetermined. It is helpful to identify drugs whichhave a high affinity for each of the subtypes ofthe receptor.

The pharmacological profile for theunknown tissue needs to be determined.

In doing this profile, choose drugs whichhave high affinities for each of the knownsubtypes. Drugs which have similaraffinities for multiple subtypes are not aseffective.

Compare the pharmacological profile for theunknown tissue with the pharmacologicalprofile of the known subtypes.

for example, suppose a drug has a high affinity forsubtype A and low affinity for subtype B and C and italso has a high affinity for the unknown receptor in the


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isolation of receptors

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