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

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 t3-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)

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).

21. 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

22.  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.

23.  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.

24. 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.

25. • 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.

26. 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

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

28. Characterization of protein recepto
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

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

30.  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

31. 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.