RECEPTOR ISOLATION, IDENTIFICATION AND CHARACTERISATION (Methods)

by Lee Eun Jin

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).

Radioligand
A radioligand is a radioactively labeled drug that can associate with a receptor, transporter, enzyme, or any site of interest. Measuring the rate and extent of binding provides information on the number of binding sites, and their affinity and accessibility for various drugs

Radioligand binding can be used to

1. Characterize receptors 2. Study receptor dynamics and localization; 3. Identify novel chemical structures that interact with receptors 4. Define ligand activity and selectivity in normal and diseased tissues.

 Receptors exist in very small concentrations in tissues. The most common method for detecting the receptors in membrane preparations, tissue slices or in the purified form is to use a radioactive drug which has a high affinity and high degree of selectivity Incubate tissue with radioactive drug under the appropriate experimental conditions, the radioactive drug (D) will bind to the receptor (R) to form a drugreceptor complex (RD).

The amount of drug-receptor complex (RD) can be measured because it is now radioactive.

Contd..
This simple model assumes; All receptors are equally accessible to ligands. Receptors are either free or bound to ligand. It doesnt allow for more than one affinity state, or state for partial binding. Binding dose not alter the ligand or receptor. Binding is reversible.

1. Identify an appropriate radioactive ligand to use for the experiment. 2. Tissue preparation to be used in the experiment 3. Identify a method for separating bound from free. 4. Identify a method for distinguishing specific from non-specific binding.

Factors to consider in designing receptor binding experiments

Criteria for selecting a radioactive ligand

a. The specific activity of the radioligand should be high enough to detect the receptor in the tissue being studied. b. The radioligand should have a high affinity for the receptor. c. The radioligand should have a high degree of selectivity for the receptor being studied. d. The radioligand should be chemically stable in the assay media during the binding reaction. e. The radioligand should be pure

Radioligand binding could be

1. Specific The site that we want to study is referred to as the SPECIFIC SITE. 2. Non-specific All other sites are called NONSPECIFIC SITE.

 SATURABLE binding sites are always present in finite amounts. Another way of thinking of this is that if you add enough ligand all of the sites will be occupied with ligand. Examples of saturable sites; Specific binding sites are always saturable All Receptors NON SATURABLE binding sites are sites that are present in essentially infinite amounts. No matter how much ligand you add, not all of the sites will be 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

SATURABLE and NON- SATURABLE binding

Theoretical Basis for Characterizing Receptors using Saturation Radioligand Assays Radioreceptor assays were first developed in the early 1970s. They were based on two very simple, but very elegant concepts.
If a ligand had high affinity for a macromolecular target (as had been shown by classical pharmacological studies over many decades), it should be thermodynamically possible to measure the binding of the ligand to the receptor without the need to perform equilibrium dialysis as long as one could separate the ligand-receptor complex from the free ligand. By labeling ligands with appropriate radioactive atoms, one could detect the ligand-

Major Types of Radioligand Study 1. Saturation binding experiments : measure

equilibrium binding of various concentrations of the radioligand. Analyze the relationship between binding and ligand concentration to determine the number of sites Bmax, and the ligand affinity, Kd. 2. Competitive binding experiments: measure equilibrium binding of a concentration of radioligand at various concentrations of an unlabeled competitor. Analyze these data to learn the affinity of the receptor for the competitor. 3. Kinetics experiments: measure binding at various times to determine the rate constants for

Saturation binding experiments

In a saturation experiment increasing concentrations of a radioactive ligand are allowed to bind to the steady-state conditions occur. After reaching steady state, the bound ligand is separated from the free ligand. The most widely used methods for separation of bound ligand from free ligand are filtration and centrifugation. The amount of ligand bound to the filter or trapped in the pellet is measured. A radioactive ligand is used because the radioactivity of low concentrations of ligand can be detected in filters or membrane pellets.

Purpose of the saturation experiment

1. To determine the affinity or Kd of a radioligand for receptor. Kd is the equilibrium dissociation of radioactive ligand required to occupy 50% of the receptors. The density (Bmax) of a specific receptor or receptor subtype in a given tissue. Bmax is the total number of receptor sites in the tissue being studied. It occurs when the all receptor molecules are occupies by radioactive drug.

1.

Methods used to determine Kd and Bmax from a saturation experiment

1. Saturation curve 2. Rosenthal plot (commonly referred to as a Scatchard plot)

1. Saturation curve
These experiments are called saturation experiments because at the higher radioligand concentrations all of the receptor molecules are occupied (saturated) by radioactive ligand. Results of the saturation experiment can be plotted with BOUND (the amount of radioactive ligand that is bound to the receptor) on the Y axis and FREE (the free concentration of radioactive ligand) on the X axis.

Contd
The resulting graph is a hyperbola and is called a saturation carve. Bmax is the maximal binding which is approached asymptotically as radioligand concentration is increased. Bmax is the density of the tissue being studied. Kd is the concentration of ligand required to occupy 50% of the binding sites.

Contd
Bmax where B is Bound B= Kd * F + and F is Free F the data to the equation for By fitting a saturation curve. Getting an accurate estimate of Kd and Bmax from this graph by eye is difficult. The curve is usually analyzed by nonlinear regression analysis.

2. Rosenthal plot
The data from the saturation experiment can be plotted with Bound/Free on Y axis and Bound on the X axis. Single site binding data can be analyzed by linear regression to give straight line. The slope of line is -1/Kd and the X-intercept is Bmax. This is a Rosenthal plot.

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 similar the slopes will be similar. If the Bmax changes then the X intercept will change. Two-site fit to the data can be visualized more easily with a Rosenthal plot than with a saturation curve.

2) Competition experiments
Many a times, ligand for receptors are not available in a radioactive form. Since they are unlabeled there is no way to directly measure their affinity for the receptor. The affinity of the unlabeled ligand for the receptor can be determined indirectly by measuring its ability to compete with a radioactive ligand for the receptor.

Contd
In a competition experiment various concentrations of an unlabeled ligand are allowed to compete with a fixed concentration of a radio labeled ligand for a receptor. As the concentration of unlabeled ligand increases, the amount of radioligand bound to the receptor decreases.

Contd
The competitive inhibitor can be either an agonist or an antagonist. It is called a competitive inhibitor because its value is determined by measuring the ability of the unlabeled drug to compete with a radio labeled drug for the receptor. The Ki for an unlabeled drug should be the same as the Kd value obtained

Purpose of competition experiments

Competition experiments can be used to determine the affinity of the unlabeled ligand for the receptor. The affinity of an agonist for a receptor. The pharmacological characteristics of subtypes of a particular receptor. The classification of receptor subtypes in a tissue.

Application of Radioligand binding assay

Methods used to identify the pharmacological profile for a specific subtype of a receptor To initial identify the pharmacological profile of a receptor subtype it is important to first have a tissue which selectively expresses only that receptor subtype. Competition studies are done to determine the 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 the receptor.

Methods used to identify the receptor subtypes in an unknown tissue

First, the pharmacological profiles for the known receptor subtypes need to be determined. It is helpful to identify drugs which have a high affinity for each of the subtypes of the receptor. The pharmacological profile for the unknown tissue needs to be determined. In doing this profile, choose drugs which have high affinities for each of the known subtypes. Drugs which have similar affinities for multiple subtypes are not as effective.

Compare the pharmacological profile for the unknown tissue with the pharmacological profile of the known subtypes. for example, suppose a drug has a high affinity for

subtype A and low affinity for subtype B and C and it also has a high affinity for the unknown receptor in the