Molecular mechanisms of GPCR Signaling for Research and Therapeutic purposes
G protein-coupled receptors (GPCRs) represent the largest family of membrane proteins. Located on the surface of cells in the plasma membrane, GPCRs bind to a wide variety of ligands, such as hormones, neurotransmitters, lipids, nucleotides, and ions, and are critical intermediaries in the transmission of signals from the outside to the inside of the cell.
They are therefore of significant importance in several therapeutic areas, such as Cardiovascular diseases, Neurosciences, Respiratory diseases, Metabolic diseases, Oncology, and Rare diseases… It has been estimated that between one-third and one-half of all marketed drugs act by binding to GPCRs (475 FDA-approved drugs target 108 GPCRs).
PerkinElmer has made available a wide range of documents with educational content (Webinar, Videos, Guide, Application notes) and reagents dedicated to GPCRs to help you in your research. We have developed a panel of ready-to-use tests to monitor second messengers, phosphoproteins, and transcription factors involved in GPCR Signaling.
The non-radioactive Tag-lite® solution has quickly risen to become the industry standard for studying receptor-ligand binding interactions.
It continues to demonstrate its effectiveness in the lab, taking the lead over traditional SPA and radioligand binding assays. Tag-lite’s success in the field is based on the many benefits it provides that are not found elsewhere:
No separation steps
No radioactive waste
Small quantities of labeled ligands
Gs, Gi, Gq Signaling
Heterotrimeric G protein complexes are composed of 3 subunits, including an α-subunit, which acts as the main signal mediator and whose active state is regulated via GTP (guanosine-5′-triphosphate) recruitment and hydrolysis, and a dimer of β- and g-subunits, which form an inseparable complex with distinct signaling abilities.
Depending on the Gα subunit involved in the complex, G-proteins are qualified as Gi, Gs, or Gq, and signal through different pathways. Gq proteins rely on enzymes of the phospholipase C family (PLC), while Gs and Gi proteins respectively stimulate and inhibit adenylate cyclase (AC) and act upon the amount of cytosolic cAMP in that way.
The PLC or AC-dependent signals are carried from the receptor to the enzymes by the Gα subunit, which is susceptible to dissociation from the receptor and its Gbg partner. In the inactive resting complex, the Gα subunit binds GDP. When the receptor is activated, conformation changes cause the GDP binding site to open, and a nucleotide exchange takes place as GDP leaves and GTP enters. This results in the separation of Gα-GTP and Gbg units from one another, and the Gα subunit transitions into an active state that dissociates from the complex to influence PLC or AC. The intrinsic GTPase activity of Gα hydrolyses bound GTP into GDP, which returns the Gα unit to its inactive state and allows it to re-associate with Gbg and a GPCR.
PerkinElmer offer a range of HTRF® and LANCE® Ultra™ kits, ensuring a match for your research needs: cAMP Gi kit, cAMP Gs dynamic kit, cAMP Gs HiRange kit, LANCE® Ultra™ cAMP kit, IP-One Gq kit, and GTP Gi Binding assay.
GTPγS – GTP Binding assay
Guanosine 5′- (trihydrogen – diphosphate), or GTPγS, is a stable analog (hydrolyzable or non-hydrolyzable) of GTP and benefits from various physiological actions, such as the stimulation of guanine nucleotide-binding proteins.
The GTP Binding assay measures G protein activation level following agonist occupation of a GPCR by determining the binding of the non-hydrolyzable analog GTPγS to the G subunits α. Thus the assay measures a functional consequence of receptor occupancy at one of the first receptor-mediated events.
This simple assay provides a better understanding of contemporary pharmacological issues, including the role of accessory proteins in signaling, GPCR receptor constitutive activity, and agonist-specific signaling.
Arrestin Signaling Pathways
Arrestins are a small family of proteins that regulate signal transduction at G protein-coupled receptors. β-arrestins play an essential role in various physiological and pathological processes, including desensitization, internalization & sequestration, and trafficking of G protein-coupled receptor vesicles (GPCR).
β-arrestin1 and β-arrestin2 are mediators of desensitization and internalization of GPCRs, and are widely distributed in various tissues and cells. β-arrestin 1 and β-arrestin 2 accumulate in the cytoplasm of cells, but β-arrestin1 also accumulates in the nucleus.
These proteins act as scaffold proteins, and are associated with certain components of the MAPK cascade and downstream targets of various GPCRs, including ERK and Akt.
Due to their ability to inhibit broad G protein signals and activate more straightforward transduction cascades, β-arrestins offer “attractive opportunities to develop therapeutics less susceptible to carrying adverse effects” than usual GPCR targeted drugs.”
For β-arrestin signaling research, PerkinElmer offer a fluorescent β-arr2 recruitment kit that allows the monitoring of endogenous β-arrestin 2 recruitment mediated by GPCR activation, and 3 HTRF kits: β-arrestin 1 total kit, β-arrestin 2 total kit, and AP2 total kit to detect the expression of endogenous or overexpressed β-arrestin 1 or 2, or AP2 in your preferred cell model.
Ligand binding induces the phosphorylation of the receptor carboxyl tail and the recruitment of one type of β-arrestin, either 1 or 2. Arrestin recruitment is followed by AP2 binding, which serves as adaptor for clathrin heavy chain recruitment. Multiple clathrins associate in a complex that coats an area around the receptor and defines it as an internalization vesicle, while dynamin seals that vesicle on the extracellular side. This process ultimately leads to the receptor internalization, which reduces the number of available receptors at the membrane and effectively desensitizes the cell.
Internalized receptors are understood to be either recycled or degraded through discrimination mechanisms that have not yet been fully described.
HTRF® Phosphorylation assays
PerkinElmer provides more than 120 ready-to-use kits for detecting endogenous and basal levels of phosphorylated proteins in a cellular context.
Cellular protein phosphorylation kits offer a rapid and easy-to-use alternative to long-standing technologies such as Western Blot, ELISA, and luminescent bead-based assays.
Find more information in the dedicated Phosphorylation from A to Z lookbook.
When GPCRs become activated, they transduce this signal through G proteins, and then a second messenger. The third part of that transduction signal chain involves phosphoprotein pathways that lead to transcription factors and changes in gene expression.
While all GPCRs differ in their specific signaling pathways, phosphatases are always involved, serving as triggers and promoters for phosphorylation cascades down to the nucleus. The MAP Kinase pathway in particular is shared by the three GPCR types we have seen, and offer additional opportunities for GPCR activity monitoring, with results in the areas of cell survival, proliferation, differentiation, and metabolism.