Receptors: Methods of studying receptors, number of receptors per cell and regulation of receptor - ePharmacology
Related topics on receptors:
- How drugs act- Different drug receptors (eg Tyrosine Kinase receptors, Hormone receptors)
- Life cycle of receptors
- G proteins
- Second messengers (cAMP, calcium, cGMP, IP3, DAG and calmodulin)
Learn about individual Receptors:
Methods of studying receptors:
The concept of receptor is not a hypothetical one. It has been possible to study the receptor by:
- Ligand binding assay
- Biochemical characterization
- Immunological characterization
- Molecular biological characterization.
Ligand Binding assay
The first step in direct characterization of a receptor is to set up an assay for specific binding. This can be done either by an agonist or an antagonist, since both bind specifically to the receptor. The receptor must be tagged in some way (most often radioisotopic labelling) that readily permits quantitation. This allows definition of affinity, receptor distribution at the tissue and subcellular level (by cell fractionation techniques), and receptor dynamic regulation (e.g. receptor internalization). Receptor autoradiography is a specialized example of receptor localization by binding assay. It may also permit recognition of receptor subtypes by differential affinity of particular ligands for different receptor preparations.
Types of ligand binding assays:
- Radioactive ligand binding assays
- Non-radioactive ligand binding assays
- Liquid phase ligand binding assays
- Solid phase ligand binding assays
Radioactive ligand binding assay
Here radioactive ligands (radioligands) are used to measure the ligands binding to receptors
Non-radioactive LIGAND BINDING ASSAY
Here a non-radioactive particle is used to measure the ligands binding to receptors. The method is similar to radioactive binding assay but the major difference is that here to radioactive toxic waste is produced. So this method the safe method of binding assay.
The techniques used for non-radioactive ligand binding assay are:
- Fluorescence polarization (FP)
- Fluorescence resonance energy transfer (FRET)
- Surface plasmon resonance (SPR)
Liquid phase ligand binding assay
The techniques used here are:
- Real-Time Polymerase Chain Reaction (RT-qPCR)
Solid PHASE LIGAND BINDING ASSAY
The techniques used here are:
- Multiwell plates assay
- On-Bead Ligand Binding assays
- On-Column Ligand Binding Assays
- Filter Assays
limitation of ligand binding assays
The limitation of ligand binding assays are they used to identify ligand binding to a receptor in a lab, i.e. outside the living body or in vitro. They cannot be used to provide receptor binding in vivo. One way of finding the ligand binding and receptor distribution in vivo is by using Positron Emission Tomography (PET). Here a radionuclide is added to a ligand. The radiolabelled ligand is then inserted into the body of the organism and then PET scan is used to locate the radiolabelled ligands. This way receptor distribution inside living organisms is studied.
Biochemical characterization of receptors
Receptor subunit composition may be determined by covalent incorporation of ligand (by photoaffinity or crosslinking techniques) and analysis on polyacrylamide gels. Receptor purification involves standard methods of protein fractionation (membrane-bound receptors must first be solubilized with appropriate detergents), as well as specialized methods such as ligand affinity chromatography. Receptor purification may allow amino acid sequencing, and immunization with purified protein to produce specific antibodies. Amino acid sequence data permits generation of peptide antibodies, and oligodeoxynucleotides for cDNA cloning.
Immunochemical characterization of receptors
Here, antibody against receptor is useful for many types of studies including immunohistochemistry, immunoblots, and definition of receptor topography (i.e. intracellular v.s. extracellular domains). Antibody may also be used in expression cloning of cDNAs.
Molecular biological characterization of receptors
Screening with antibody may permit cloning of cDNAs encoding receptors. Cloning of receptor cDNAs and genes immediately provides a wealth of data including the primary structure.
Number of receptors in a cell?
It has been possible to count the number of receptors per cell. The number of receptors in different tissues varies.
The neuromuscular junction in skeletal muscle contains high density of nicotinic cholinergic receptors (10,000 per square micrometer).
There are about 85,000 β-adrenergic receptors per cell in dog ventricular muscle.
Erythrocytes contains only 40 insulin receptors whereas hepatocytes contains about 300,000 insulin receptors per cell.
Hemoglobin synthesizing reticulocytes have approximately 300,000 transferrin receptors on the cell surface.
The density of acetylcholine receptors increases after chronic exposure to nicotine but is decreased in patients with Alzheimer’s or Parkinson’s disease.
Receptors are dynamic rather than static entities. Receptor number and affinity can both be altered by a variety of mechanisms. A major factor regulating receptor function is the exposure to agonist. Prolong removal of agonist (as in denervation or prolonged treatment with antagonist) can lead to receptor supersensitivity. Prolonged exposure to agonist may lead to desensitization, i.e. reduced response despite continued exposure to bioactive agonist.
Desensitization has been divided into homologous and heterologous types. In the homologous type, reduced response is evident only for specific receptor that has been exposed to agonist. In the heterologous type, response is reduced in receptors for other agonists.
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