Proteins receptors (Identification and Characterization)

Proteins receptors (Identification and Characterization)

1. PROTEIN RECEPTORS(IDENTIFICATION AND CHARACTERIZATION) SUBJECT: PROTEOMICS

2. Proteins receptors Receptors are proteins or glycoprotein that bind signaling molecules known as first messengers, or ligands. They can initiate a signaling cascade, or chemical response, that induces cell growth, division, and death or opens membrane channels. A receptor protein is meant to recognize and bind to specific substances outside of the cell

3. LOCATION OF PROTEINS receptors Receptor proteins  are located: Within the cell surface membrane, Nucleus membrane cellular organelle membrane.

4. FUNCTIONS OF PROTEIN receptors Receptors are bound up with functions such as  cell activation   Immune system T-cell, B-cell, NK-cells cell adhesion    "Sticky" molecules(AR’S) Migrate, proliferate, and survival (AR CLASSES) Integrins, cadherins, selectins, and immunoglobulin-like cell adhesion molecules (Ig-cam) signaling pathways e.g ELR, GPLR, ICLR

5. Types of protein receptors There are two types of receptors: internal receptors (Intracellular or cytoplasmic receptors, are found in the cytoplasm of the cell and respond to hydrophobic ligand molecules that are able to travel across the plasma membrane) cell-surface receptors ( Cell-surface receptors, also known as transmembrane receptors, are cell surface, membrane-anchored, or integral proteins that bind to external ligand molecules )

6. A. INTRACELLULAR RECEPTORS Intracellular or cytoplasmic receptors Found in cytoplasm of the cell Responds to hydrophobic ligand molecules They are regulators of mRNA synthesis

7. Types of intracellular receptor proteins Thyroid and steroid hormones receptors(transcriptional factors, nuclear receptors) IP3 receptor located on endoplasmic reticulum. Sigma1 Intracrine peptide hormone receptors

8. 1. THYROID HORMONES RECEPTOR Thyroid hormone is signaled by the cell through nuclear  thyroid hormone receptors  ( trs ). TRs are members of the so-called  nuclear receptor  superfamily Influence gene expression by binding to specific DNA elements as  dimers TR can bind as a  homodimer  (two identical monomers) or as a  heterodimer  (two different monomers) to these specific DNA elements, called thyroid  response elements  ( tres ), located in the promoter region of t 3 -responsive genes Unique among their family (influence gene expression whether bound by ligand or not this is the result of the fact that the TR can bind to a TRE without hormone)

9. THYROID HORMONES RECEPTOR

10. 2. STEROID HORMONES RECEPTORS

11. B. cell-surface receptors  Receptors that are embedded in the plasma membrane of cells They act in cell signaling by receiving (binding to) extracellular molecules They are specialized integral membrane proteins that allow communication between the cell and the extracellular space Extracellular molecules may be hormones, neurotransmitters, cytokines, growth factors

12. TYPES OF CELL surface RECEPTOR PROTIENS Membrane receptors are mainly divided by structure and function into 3 classes: The ion channel linked receptor The enzyme-linked receptor And the G protein-coupled receptor.

13. IDENTIFICATION of protein receptor There are a lot of interesting ways one can go about identifying a receptor for a particular ligand or set of ligands, but the best approach usually depends on the biology/biochemistry of your protein receptor and the available ligands. There are two general types of approaches you can take to do this: Approach 1 : the biochemical approach Approach 2 : the molecular biology and/or genetics approach

14. For most of the major g-protein couples receptors  Is to use biochemistry and pharmacology to chemically isolate the receptor from a native source, and identify it by  mass spectrometry or edman degradation. For example: isolation of sigma-2 receptor Approach 1 : The biochemical approach

15. The sigma-2 receptor binding site was defined as an 18–21.5 kda membrane protein that had high affinity for  ditolylguanidine  (DTG) and haloperidol, but not benzomorphans (in contrast to the sigma-1 receptor, which binds all of these ligands with high affinity). Many ligands have been developed over the years, but we didn’t know what gene actually coded for the receptor, which made it’s study difficult. 

16. STEPS Identify a source of your receptor: First, we needed to identify a biological source of the receptor we could use for study. The sigma-2 receptor was known to be expressed at relatively high levels in pc-12 and mcf-7 cell lines. A classical source of sigma-2 receptor were cell membranes isolated from rat or pig liver. Tests showed that per mg of membranes, calf liver membranes had the same amount of sigma-2 receptor binding as rat liver membranes. So, we can use a bunch of calf livers. 

17. Extract the receptor from its source : Now grind up the liver in a blender and spin the homogenate at high speeds to isolate the membranes, which come out of solution. Then, wash them several times to remove as many soluble proteins as possible. Now we had cell membranes with the receptors, we could use a detergent, to extract most of the membrane proteins from the membranes. Throughout this process, we used radioactive (DTG) material to detect the receptor, to make sure we carried it through each step.

18. Leverage existing chemistry to enrich for your receptor in your sample: In principle, we could have done a series of biochemical fractionation steps to enrich for our protein of interest. Prepare ligands for receptor Fixed it to a column and washed the detergent-solubilized liver membranes over it. Then, we added DTG to out-compete the ligand on the column. This didn’t make the receptor perfectly pure, but it did enrich for it to a great degree. We ran the sample on a silver stain gel, and then cut out the band that was in the 18–21.5 kda range.

19. Get a list of candidate genes: This band was sent for mass spectrometry, and we got a list of proteins back. We picked the most interesting ones and expressed those recombinantly in human HEK 293 cells, and tested them to see if they bound radioactive DTG in a way that could be blocked by non-radioactive haloperidol (since sigma-2 bound both, it should bind the radioactive DTG, but this should be able to be competed off by cold haloperidol).

20. Validation: Only one of the genes tested produced a protein with the properties described above: tmem97 We followed up with some validation, mainly showing that sigma-2 ligands bound to TMEM97 with the same affinity as they did for sigma-2

21. BENEFITS The direct biochemical approach has probably the highest success rate for identifying and isolating receptors of interest when you have no idea what the gene might be, largely because it is a generalizable method that doesn’t rely on knowing anything about the genetics or molecular biology of your system until the validation step. You just have to be able to identify a source of your receptor and extract it using the right combination of biochemical methods.

22. LIMITATIONS: There are some limitations. The main one is that you need a way to reliably and easily track the binding of your ligand to your receptor of interest throughout the purification. Otherwise, you will not be able to isolate the receptor because you will have no idea which biochemical fraction it is in. In most cases you would use radioactive ligands, but fluorescence would probably work too.

23. Approach 2: The molecular biology and/or genetics approach This class of approaches have the potential to be a lot faster than the biochemical approach, but it requires more knowledge of your system and for the right tools to be available. Basically, the idea with these approaches is to skip isolating your receptor, and just get a list of candidate genes by exploiting either the biology of your receptor or the ability to screen single cells for binding with techniques like ( Flourescence -activated cell sorting) FACS.

24. The simplest version of this type of approach is to use known sequence information to amplify cDNA from your source that may have the properties you want Having a ligand of interest that can bound to a certain cell type or tissue, and you thought you knew what kind of sequence the receptor for that ligand  should  have, then you could amplify those cDNAs, express them in cells, and screen for binding. If you had a fluorescent ligand and thought your receptor may be able to be expressed in yeast, then you could even amplify all cDNA from your source tissue, and then transform this cDNA library into yeast. Then, you could use FACS to sort out fluorescent yeast, and see what cDNAs were associated with binding to your fluorescent ligand.

25. DRAWBACK This has the potential to be a very fast method, however, it is somewhat less generalizable because you need to either have a fluorescent ligand, and/or some idea of what your receptor looks like already

26. Deferent methods for identification Radioligand binding assay Flourescent ligand binding assay Competative binding assay Receptor binding assay

27. Characterization of protein receptors Characterization : a description of the distinctive nature or features of someone or something The genome encodes a wide range of Protein receptor but the function of most of these proteins is unknown. its necessary to find their function such as for pharmacological purposes. So we use different methods of characterization to illustrate the nature and features of protein receptors

28. CHARATCERIZATION METHODS OF PROTEIN RECEPTORS On the basis of pharmacological responses Radio ligand binding studies Molecular cloning techniques Analysis of biochemical pathways linked with receptor activation

29. Radio ligand binding studies Radio ligand studies are helpful in: Characterize receptors in their natural envirnment Study protein receptor dynamics and localization Interaction of chemical structures with receptors Defines ligand activity and selectivity

30. Receptor pharmacological studies The study of the interactions of protein receptors with drugs/pharmaceuticals and other xenobiotics. A basic tenet of receptor pharmacology is that a drug's effect is directly proportional to the number of occupied receptors.

31. Thank you