مستقبل G المرتبط بالبروتين: من إشارات الخلايا إلى اكتشاف الدواء

G protein-coupled receptors, or GPCRs for short, are membrane proteins which assist cells respond to signals from outside the cells. These signals may encompass hormones, neurotransmitters, lipids, peptides, as well as light.

While there is a clear commonality of structure in the seven-transmembrane structure of the GPCRs, they do not function in the same manner. The results of an experiment may depend on the ligand for the receptor, the cell's environment, the level of expression of the receptor, and the signaling partners.

Researchers, then, have to go beyond this measurement of a compound's "activation" of a receptor to decipher GPCR biology. All the pathway, timing, assay format, controls and cell model are important.

G Protein Coupled Receptor: What is it?

The number of times a typical GPCR will traverse the cell membrane is 7. The extracellular portions of it bind to signaling molecules and the intracellular portions bind to the G proteins, arrestins, kinases and other signaling molecules.

GPCRs are classified into a number of major classes by the IUPHAR/BPS Guide to Pharmacology, such as Class A, Class B, Class C, Frizzled and Adhesion receptors. Class A is the largest, and involves well known targets, like the adrenergic, the dopamine, the opioid and the muscarinic receptors.

Although similar in structure, GPCRs are able to recognize a very diverse set of signals. This enables them to engage in sensory perception, cardiovascular regulation, metabolism, immune functions, mood, movement and so much more.

How Do GPCRs get Activated?

A ligand binds to the receptor, and causes the receptor to change shape. As a result of this conformational change, the receptor is able to interact with an intracellular signaling protein.

Many GPCRs have a heterotrimeric G protein as the first partner composed of alpha, beta and gamma subunits. Activation of the receptors results in the exchange of GDP for GTP in the alpha subunit. The activated components then affect the downstream enzymes, ion channels and second messengers.

The most popular families of G proteins are:

• Gs, that typically causes an increase in cyclic AMP
• The commonly used name for the cyclic AMP-reducing enzyme is gi/o.
• The Gq/11 pathway that signals phospholipase C and calcium pathways
• The Rho-family protein and cytoskeletal responses can be controlled by G12/13.

This textbook description is helpful, but a line is a rare form of signaling! A single receptor can signal through multiple G proteins and the optimal signal transduction can vary depending on ligand and cell type. GPCRdb mentions that receptors can interact with G proteins, GPCR kinases and arrestins, which lead to formation of a variety of intracellular signalling pathways.

The roles of arrestins

Once the G protein has been activated, the signaling by GPCRs does not stop.

GPCR kinases are able to phosphorylate activated receptors. Arrestins could then interact with the receptor, inhibit further coupling of the receptor to G proteins, facilitate receptor internalization and be involved in other pathways of signal transduction.

Therefore, the recruitment of arrestin cannot be considered as an “off switch.” Depending on the receptor and experiment it can affect the trafficking, desensitization, recycling, degradation of the receptor or modulate other downstream effects.

Since both G protein signaling and arrestin recruitment are important measures of the complete pharmacology of a compound, they are frequently measured together.

Articles in this section examine the ways in which the same receptor can lead to different responses

The GPCRs are not just on/off switches.

Various ligands can be found to bind to different conformational phases of the receptor. One type of ligand can cause high levels of activation on one G protein pathway vs. the other pathway can cause more arrestin recruitment or different signaling profile. It is typically referred to as functional selectivity/biased signaling.

Backgrounds of the cells are also important. The same receptor can be found in two different labs, but have different amounts of G proteins, arrestins, kinases, enzymes, or endogenous receptors in the cells.

Another problem may be the level of the expression of receptors. Excessive expression can enhance the responses to low levels of activity and/or alter coupling preferences, and can also lead to a partial agonist having stronger activity in the cell than in native tissue.

A result should therefore be taken as an indication of the behaviour of a complete experimental system and not solely of the receptor.

Select Assay based on biological question

No one GPCR assay can answer all the questions.

cAMP Assays

Receptors with Gs or Gi proteins typically are measured using cAMP. A Gs response could lead to an increase in cAMP and a Gi response could be indicated by the inhibition of stimulated cAMP production.

These assays are commonly used in concentration-response studies, in characterization studies of agonists and in antagonist studies. In antagonist experiments, a concentration of the agonist is typically applied after the exposure of the test compound to the cells.

Calcium Assays

Gq-coupled receptors may be studied by intracellular assays of calcium. They can give a quick and high throughput readout, but the response might be short in duration, and highly dependent on the handling of the cells, loading of the dyes, receptor expression, and time of the measurement.

GTPγS Binding

GTPγS assays are one of the earliest events to be measured in the process of G protein activation and they are often used to confirm the activity of the agonist in membrane preparations. Since the assay is rather near to the receptor–G protein interaction, it can lessen a few of the amplification experienced in downstream readouts.

Arrestin Recruitment

Arrestin assays can be used to investigate pathway bias, ligand-specific signal, receptor trafficking and receptor desensitization. They are best used in conjunction with a G protein assay and not as a general indicator of the activation of a receptor.

Live-Cell Biosensors

BRET and FRET techniques (and other biosensor methods) can track signaling events in living cells as a function of time. The biggest selling point of their is the kinetic information: researchers are able to measure when signaling starts, when it peaks and then falls, since they measure more than one endpoint.

Make an accurate GPCR Experiment

A good assay is based on the biologic and not the instrument.

To begin with, make sure that the receptor is expressed and is situated at the correct location in the cell membrane. The low response may be not a result of the low activity of the ligand, but a result of poor receptor expression/trafficking.

Secondly, add appropriate control devices. Commonly, a known agonist, a known antagonist, vehicle controls, untransfected cells and a receptor-independent control for the downstream assay may be needed.

Finally, perform a complete concentration-response curve. An evaluation of only one concentration of a compound will not be a reliable measure of potency, efficacy, partial agonism, toxicity, or non-specific interference with an assay.

Standardization of the timing is also needed. Once you get a calcium signal, within a few seconds, and then a few hours later you measure gene expression, you can't simply compare the two. Tests measuring the same receptor can be used to measure different phases of the response.

Lastly, validate significant results using orthogonal approach. If a compound is found to be active in a cAMP assay, it is best to confirm it with another useful assay, binding assay or with a receptor specific positive control. The Assay Guidance Manual suggests that the careful validation, quality control and pharmacological characterization of GPCR hits in moving toward lead optimization will be recommended.

GPCRs for Drug Discovery

GPCRs continue to be a key role in drug discovery as they are involved in many aspects of physiology and are surface-expressed on the cell. GPCRdb estimates that 25% of human hormones are involved in response to members of this receptor family, and that approximately 1/3 of all drugs target members of this receptor family.

For traditional programs, the main search targets were either agonists or antagonists that bind to the main ligand-binding site. Investigation of allosteric modulators, pathway-biased ligands, receptor – transducer selectivity, binding kinetics and structure-based compound design are also currently under investigation.

With the advent of structural biology, this has become more feasible. GPCRdb now includes tools to compare binding sites and activation states of GPCRs, including experimentally determined receptor structures, receptor–ligand complexes and receptor–G protein complexes.

A compound with good structures should be subjected to functional test even in having good structures. A molecule can bind to an apt target with strong binding affinity and not trigger the intended cellular response or alternatively, a molecule could engage the desired pathway but not in the correct manner.

استنتاج

The G protein-coupled receptors are best thought of as not just a membrane switch, but a flexible signaling system.

The receptor changes shape in response to a ligand binding, the receptor finds its partners in the cell and the environment of the cell dictates the course of the signal. That's why it is important to consider the choice of assays, the expression of the receptors, the timing of the pathway, controls and orthogonal validation.

The best conclusions for GPCR research and drug discovery are often reached as a result of a combination of structural information, and more than one functional assay.

الأسئلة الشائعة

س1. What Is the Main Function of a GPCR?

A GPCR is a cell surface receptor that responds to an extracellular signal and produces an intracellular response. The answer could be via G proteins, arrestins, second messengers, ion channels or gene regulation.

س2. Are all GPCRs activated by the same G protein?

No. Various receptors can prefer to activate various G proteins (Gs, Gi/o, Gq/11, G12/13 or multiple signalling partner(s)).

س3. What is an Orphan GPCR?

An orphan GPCR is a receptor for which the natural activating ligand is not certain. These receptors still are of major significance to basic research and drug development.

س4. So, which Assay to Use for GPCR Screening?

The "best" assay will depend on the question being addressed and the signaling pathway of the receptor. Different aspects of the response can be measured by cAMP, calcium, GTPγS, arrestin and live-cell biosensor assays.

س5. What is the reason for researchers to employ more than one assay?

One assay can measure only one pathway or have format specific interferences. If it can be demonstrated with an orthogonal assay that the effect observed is indeed receptor-related, then it is possible to conclude that this is truly an effect of the receptor.