cell signaling mechanism

1. METHODOLOGY OF PURIFICATION,
RECONSTITUTION AND CHARACTERIZATION OF
RECEPTORS, SYNTHETIC ANALOGUES OF
EPINEPHRINE
Presentation by
DEBASHISH CHAKRABARTY
M.Sc-3rd Sem
SLS

2. NEED FOR ISOLATION AND RECONSTITUTION
• Receptor proteins are present in low levels in
biological system.
• Lipid membrane is complex, heterogeneous and
dynamic environment so limits the use of NMR,
X-ray diffraction etc.
• Receptor proteins are trans membrane proteins
so can’t be studied in aqueous medium.
• Easy manipulation in reconstituted system.

3. ISOLATION OF RECEPTORS
- Receptor Sources
Cloning
- 2 methods
Natural source
• Cloning
Gene for the receptor Inserted within an expression vector
Screening Protein Expression Host Transformation
 Expression is done by synthesizing fusion protein where tags are
used.

4. Cell Disruption
• Yields a suspension of membrane fragments along with other sub
cellular structures.
Methods used are:-
• Ultrasonication.
• Glass bead milling.
• Osmotic Shock.
 Followed by differential centrifugation.

5. Solubilisation
• The process where membrane proteins are extracted from the
lipid membrane by using detergent.
• Protein detergent complexes are formed and stabilize the
proteins.
Purification
• The process to obtain only the desired proteins.
• All the steps are carried out in presence of detergents.
• Combination of 2 or 3 purification methods are used.
.

6. • Affinity purification(eg. Histidine tagged
proteins)
- Based upon the affinity of the desired protein towards
a particular molecule.
• Ion Exchange Chromatography
- Based upon the charge of the desired protein.
• Gel filtration Chromatography
- Based upon the size of the protein.

7. RECONSTITUTION OF RECEPTORS
Detergent and mixed micelles
-Most basic strategy.
-Prepared from mixture of detergents.
Drawbacks
• Presence of high conc. of free detergent.
• Micelles are much more disordered.
Bicelles
• Detergent Solubilised receptors are mixed with short and long
chain lipids.
• Lipid bilayer is formed; perimeter stabilised by short chain lipids.

8. Advantages
• Diameter allows multiple receptor reconstitution.
• Reduced free detergent concentration.
Drawbacks
• Presence of empty bicelles.
Nanodiscs
• Most novel paradigm for GPCR.
• Resemble bicelles.
• Consists of lipid bilayer and MSP.

9. Advantages
• No high detergent concentration.
• MSP provide additional labelling sites.
• Don’t have enclosed topography.
Disadvantages
• No difference in extracellular and intracellular
environments.
• Constraints on the diffusion of receptor proteins.

10. CHARACTERIZATION OF RECEPTORS
• Most studied receptor is β adrenergic receptor.
PRIMARY STRUCTURE
• Single polypeptide chain.
• 400-500 amino acids long.
• N terminus and C-terminus; 3 extracellular(e), 3
intracellular(i) stretches and 7 transmembrane domains.

11. -modified from Kobilka et al. [1987]

12. Post Translational Modifications
1. N-linked glycosylation
• 1 or 2 sites within the N-terminus.
• Absence doesn’t alter ligand binding or signal transduction.
2. Palmitoylation
• Occurs at a Cys residue present immediately after tm7 domain.
• Helps in mediating agonist resposne.
3. Disulfide bond
• Cys 106 and Cys 184 residue involved in disulfide bonding.

13. Ligand Binding Site
• A pocket within the transmembrane is the place for
ligand binding.
• Asp 113 in tm3 while Ser 204 and 207 in tm5 are crucial
for ligand binding.
-Strosberg, 1991b

14. SIGNAL TRANSMISSION
• Asp 79 residue present in tm2 is responsible for binding of
agonists.
• Asp 79 and Tyr 316 helps in activation of G protein.
• Tyr 316 and Asn 312 is prsent in tm7 prevents activation of G
protein(antagonists).
G PROTEIN INTERACTION
• I3 loop is the main site that interacts with G protein.
• I2 also has some contribution.
• Homologus replacement or deletion of the sequence is used for
studies.

15. -Strosberg et al., 1993

16. SYNTHETIC ANALOGUES OF EPINEPHRINE
• Epinephrine is a hormone secreted by adrenal medulla
and binds to the adrenergic receptors.
• Synthetic analogues are chemically synthesized which
can mimic its action e.g- Isoproterenol.
• Epinephrine is a catecholamine so an analogue must
contain some of the groups that can interact with the
receptor :-
N-methyl group
-Packs against Phe 411 and Phe 412 in tm7.

17. Epinephrine
Isoproterenol
(Analogue) -From wikipedia
-From wikipedia

18. Β-OH group
-Forms Hydrogen bond with Asp 113.
Aromatic group
-Inserted within the tm6 and interaction takes place with
Phe 391 and Phe 394.
Para and Meta OH groups
-Contacts residues within tm3 and tm5. eg.- Thr 118 in
tm3 and Ser 204 in tm5.

19. IN BRIEF
• Receptor proteins can’t be studied within native
sites.
• Needed to be isolated and purified in order to
reconstitute in environment that mimics the
native site.
• Characterization helps in studying
conformational changes of receptors during
ligand interactions.
• The synthetic analogues can be designed on the
basis of ligand structure and receptor
characterization.

20. References:-
• GE health care- purifying challenging proteins.
• Functional reconstitution of β2-adrenergic receptors utilizing
self-assembling Nanodisc technology;Andrew J. Leitz1, Timothy
H. Bayburt1, Alexander N. Barnakov2, Barry A. Springer2, and
Stephen G. Sligar.
• Structure, function, and regulation of adrenergic receptors; A.D.
STROSBERG Laboratoire d’lmmuno-Pharrnacologie Moleculaire,
lnstitut Cochin de Genetique Moltculaire, and Universite de Paris
VII, Paris, France.
• Artificial membrane-like environments for in vitro studies of
purified G-protein coupled receptors ;Eugene Serebryany, Gefei
Alex Zhu, Elsa C.Y. Yan.

21. THANK YOU