Receptor down regulation

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) [email protected] 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 P resent 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 U ses storage phosphor screens and lasers to detect radioactivity G enerates 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 .