Receptor Down-regulation

Chander K NegiM.S (Pharm)

chandernegi09@gmail.com

Department of Pharmacology and Toxicology

National Institute of Pharmaceutical Education and Research (NIPER)

Sector-67, S.A.S. Nagar, Mohali, Punjab-160062

Receptor

A protein molecule Present either in plasma membrane or cytoplasm Molecule bind to receptor termed as ligand It may be peptide, neurotransmitter, hormone,

drug or toxins Ligand may be agonist or antagonists

Regulation of receptor

Two different method for regulation of receptor

Up-regulation Down-regulation

Cell increase the quantity of cellular component

Cell decrease the quantity of cellular componentIn response to external variable

Receptor down regulation

It is the response of cell for continuous exposure of ligand It is the reversible process Receptor down-regulation is the result of various cellular

processes including receptor internalization, new synthesis, and recycling

Most membrane receptors are internalized in the receptor mediated endocytosis

For some receptors internalization requires ligand binding to receptor and for others ligand binding is not necessary

Receptor down regulation

Degradation of receptor (protein) occurs in two ways

Lysosomal degradationoccur via lysosomes

Non-lysosomal degradationoccur via proteosome

(ubiquitin)

This step occurs in downregulation of receptor

Mechanism of Receptor down regulation

TECHNIQUES FOR DETERMINATION OF DOWNREGULATION

IMMUNOFLUORESCENCEPHOSPHORIMAGINGAUTORADIOGRAPHY

IMMUNOFLUORESCENCE

Immunofluorescence is the labeling of antibodies or antigens with fluorescent dyesDirect immunofluorescenceIndirect immunofluorescence

It is detect location and abundance of any protein. For immunofluorescence, we need antibody against

particular receptor (protein) Example : GPCR

Direct Immunofluorescence

Indirect Immunofluorescence

PHOSPHORIMAGING PHOSPHOR + IMAGING PHOSPHOR means a substance that exhibits a phenomenon

of PHOSPHORESCENCE, sustained release of light after exposure to energized particles such as electrons or UV.

A phosphorimaging is based on imaging plates. Imaging plates consists of a thin layer of special crystal

doped with a lanthanides. A quantitative (sensitive) imaging technique Uses storage phosphor screens and lasers to detect

radioactivity Generates images similar to autoradiographs

• Take a sample which is previously hybridized with labeled probe ( P 32)

• It is placed in contact with phosphor image plate on specimen microscope slide

• When we apply radiation to the sample it will excites sample molecules on the plates

• This molecules remain in excited state until the phosphor imager scans the plate with laser, it produce latent image

beta rays energy trapped by plate is released in the form of visible light

And this visible light is monitored by computerized detector

A false color image produced where the different color represent different of radioactivity from lowest (yellow) to highest (black)

AUTORADIOGRAPHY

X-ray film basedUsed radioactive labeled molecule or

sampleRecording medium : photographic

emulsion

Living cells are briefly exposed to a ‘pulse’ of a specific radioactive compound.

The tissue is left for a variable time. Samples are taken, fixed, and processed for light or electron

microscopy. Sections are cut and overlaid with a thin film of photographic

emulsion. Left in the dark for days or weeks (while the radioisotope

decays). This exposure time depends on the activity of the isotope, the temperature and the background radiation (this will produce with time a contaminating increase in ‘background’ silver grains in the film).

The photographic emulsion is developed (as for conventional photography).

Counterstaining e.g. with toluidine blue, shows the histological details of the tissue. The staining must be able to penetrate, but not have an adverse affect on the emulsion

Alternatively, pre-staining of the entire block of tissue can be done (e.g. with Osmium on plastic sections coated with stripping film [or dipping emulsion] as in papers by McGeachie and Grounds) before exposure to the photographic emulsion. This avoids the need for individually (post-) staining each slide.