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

1. Receptor Down-regulation
Chander K Negi
M.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

2. 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

3. 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 component
In response to external variable

4. 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

5. Receptor down regulation
Degradation of receptor (protein) occurs in two
ways
Lysosomal degradation
occur via lysosomes
Non-lysosomal degradation
occur via proteosome
(ubiquitin)

6. This step occurs in downregulation of receptor
Mechanism of Receptor down regulation

7. TECHNIQUES FOR DETERMINATION
OF DOWNREGULATION
IMMUNOFLUORESCENCE
PHOSPHORIMAGING
AUTORADIOGRAPHY

8. IMMUNOFLUORESCENCE
 Immunofluorescence is the labeling of antibodies or
antigens with fluorescent dyes
Direct immunofluorescence
Indirect immunofluorescence
 It is detect location and abundance of any protein.
 For immunofluorescence, we need antibody against
particular receptor (protein)
 Example : GPCR

9. Direct Immunofluorescence

10. Indirect Immunofluorescence

11. 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

12. • 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

13.  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)

14. AUTORADIOGRAPHY
 X-ray film based
 Used radioactive labeled molecule or
sample
 Recording medium : photographic
emulsion

15.  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).

16.  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.