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1. "Mutational Studies to Identify Residues Involved in the Internalization of the Rat 5-HT_2A Receptor."

National Center for Biological Sciences (Tata Institute of Fundamental research), Bangalore, India.

Guide: Dr. Mitradas M. Panicker

Background of the project

As 5-HT2A receptors activate the phospholipase-C pathway, the effect of protein kinase C (PKC) on the internalization process of the receptor was studied.

A construct was created in which the GFP was tagged to the C terminus of the 5HT_2A receptor for easy visualization. The receptor remains active and behaves as the native receptor, when tagged with GFP and the fusion receptor internalizes on exposure to serotonin, its native ligand .  Activation of PKC by its specific activator PMA (phorbol 12-myristate 13-acetate) causes internalization of the 5-HT_2A receptor in the absence of the ligand serotonin showing that activation of PKC is sufficient to cause internalization. Inhibition of PKC by its inhibitor sphingosine in the presence of 5-HT prevents the internalization process, thereby, suggesting that activation of PKC is not only sufficient but also necessary for the internalization of 5-HT_2A receptors. 

Ligand-independent internalization of the 5-HT2A GFP receptor upon activation of PKC by PMA in HEK293 cell line

                Control                         10nM PMA                         1mM PMA

Inhibition of the 5-HT2A GFP receptor internalization in the presence of sphingosine (an inhibitor of PKC) in HEK293 cell line.

          Control                    10mM 5-HT                10mM 5-HT+50mM       10mM 5-HT+200mM

Scheme of the experiment and Results

In order to identify the molecular mechanisms behind the process, I had checked the role of the residues present in the third intracellular loop and at the C terminus of 5-HT2A receptors, which might be phosphorylated by PKC and thus played a role in the internalization process. These residues were mutated sequentially by site-directed mutagenesis. Failure to internalize implied that the residue must be involved in the internalization of the receptor.   

Discussion

Serotonin-2A (5-HT_2A) receptors are seven-transmembrane G-protein coupled receptors (GPCRs).  The sequence of the third intracellular loop has been found to be conserved (98% homology) between rat and human 5HT_2A receptor, suggesting a strong conservation of G-protein coupling.

Various antipsychotic agents and antidepressants bind with relatively high affinity at 5-HT_2A receptors. Chronic administration of 5-HT_2A antagonists results in a paradoxical down-regulation of 5-HT_2A receptors; such a down-regulation would be of benefit in the treatment of depression.  Antipsychotic drugs such as clozapine, olanzapine, risperidone, sertindole, and ziprasidone that are potent serotonin 5-HT_2A antagonist, as well as being a slightly less dopamine D₂ antagonist, (serotonin-dopamine antagonist concept) are very effective against diseases like schizophrenia. These drugs show lesser extra pyramidal side effects (EPS) than those, which are more potent at Dopamine receptors

As the vasoconstrictor effects of 5-HT_2A receptor stimulation are markedly potentiated in hypertension and atherosclerosis, selective 5-HT_2A receptor antagonists, notably ketanserin, are also clinically useful for hypertension.

Therefore, any findings from these experiments regarding the internalization mechanism of 5-HT_2A receptors would be an important information to design drugs that act through this receptor. 

(The project has now been completed and the residue which is responsible for the internalization process has been identified. The results will be sent for publication.)

2. "Identification of Anti-Inflammatory Molecules that Block the Leukotriene Biosynthetic Pathway, Towards the Development of a Drug for the Cure of Asthma."

Cellular Biochemistry Division, Indian Institute of Chemical Biology, Kolkata (Under CSIR, Govt. of India)

Guide: Dr. Siddhartha Roy & Dr. Ranjan Bhadra

The oxidation of arachidonic acid is mediated by 5-lipoxygenase leading to the formation of leukotrienes, which are powerful triggers to evoke asthma. Therefore the blocking of this enzyme is being targeted as a therapeutic strategy for the control of asthma. As part of an interdepartmental and inter-institutional project, I am screening different compounds of synthetic and natural origin, to identify potential inhibitors of this enzyme, which will eventually lead to the development of a drug for asthma.

(The project is in progress.)

3. "Study of Immunomodulatory Responses on Macrophage Cells by Leishmanial Lipoproteins."

Cellular Biochemistry Division, Indian Institute of Chemical Biology, Kolkata (Under CSIR, Govt. of India)

Guide: Dr. Ranjan Bhadra & Dr. Krishna Das Saha

Lipoproteins from several bacteria, Mycobacteria and Mycoplasma show profound immunomodulatory roles including cellular apoptosis. Lipid from Leishmania sp. has been seen to modulate the function of the immune system. This lipid has been found to be tightly associated with some proteins. Since, macrophage is the host of Leishmania parasite, the present study was designed to venture the role of Leishmanial lipoproteins on immune responses of the macrophage cells. Effects on cytokines (interleukins and TNF-a), induced nitric oxide, ROS and signaling intermediates (PKC, MAPK and NF-kb) are being studied by FACS, Western-blot analysis, RT-PCR and ELISA studies.

(The project is in progress.)