Internalization Assay Services

PCRs are the main therapeutic target in drug discovery, nearly half of the drugs currently in clinical use target G-protein-coupled receptors (GPCRs). GPCRs are involved in a broad diversity of physiological functions, such as pain perception, chemotaxis, neurotransmission, cardiovascular actions, and metabolism, and finding ways to modulate GPCR signaling remains a major focus of pharmaceutical research. Creative Biolabs’ team of highly qualified and experienced technical staff will work with you to develop and deliver GPCR internalization assay to advance your drug discovery projects.

Overview of GPCR Internalization Assay

GPCRs are seven-transmembrane helix proteins, typically transmitting an extracellular signal into the cell by the conformational rearrangement of their helices and by the subsequent binding and activation of an intracellular heterotrimeric G protein. The functional activation of GPCRs may be monitored by agonist-induced receptor internalization. The broad applicability of GPCR internalization assays is based on the common phenomenon of GPCR “desensitization”. The internalized GPCRs can continue to either stimulate or inhibit cAMP production in a sustained manner. In addition, internalization is involved in the ligand-clearing function of atypical GPCRs, which circulate between the plasma membrane and the intracellular lumen. The internalization contributes to the diversity of GPCR-dependent signaling pathways and its dynamics in living cells have generated considerable interest.

Scheme of GPCR Internalization Assays

In the desensitization process, GPCR-kinases (GRKs) phosphorylate agonist-activated GPCRs on serine and threonine residues. Arrestins are cytoplasmic proteins that are recruited to the plasma membrane by GRK-phosphorylated GPCRs. Arrestins then uncouple the GPCR from the cognate G protein and target the desensitized receptors to clathrincoated pits for endocytosis. The internalization process can be monitored by fluorophore labeling on agonists, N-terminus, or C-terminus of GPCR, or statins.

Fig.1 Scheme of GPCR internalization assays. (Haasen, 2006)

Internalization Assay Protocols

One means to monitor the internalization of a GPCR is to cointernalize a specific, fluorophore-labeled ligand. In the following example protocol tetramethylrhodamine-labeled endothelin-1 (TMR-ET-1) is used as a ligand for the endothelin A receptor (ET_AR).

1. Seed cells into assay plate using a multichannel pipette at a density of 25,000 cells in a volume of 100 μL volume per well in complete medium. Culture for 20 to 24 h at 37℃, 5% CO₂.
2. Remove the complete medium and add 40 μL of ligand in HBSS/HEPES.
3. Incubate assay plate at room temperature.
4. Add 1 volume 2×concentrated fixing solution to wells at indicated time points.
5. Fix for 30 min at room temperature.
6. Remove the fixing solution and add 100 μL PBS.

Advantages of The Internalization Assay

The internalization assay compares favorably to the arrestin-based activation assay and therefore should be useful in general, as well as specifically for assaying arrestin-independent GPCRs. The internalization assay is specific since it can be inhibited by antagonists and low temperatures.

Receptor internalization is a universal process and an indicator of activation of many GPCRs, including orphan receptors. Benefit from working with a one-stop shop by partnering with Creative Biolabs. We do not just provide the results, pass, or fail, we work to understand the results, and if they do not meet your quality criteria, we can help with troubleshooting to ensure they will. Please feel free to contact us to discuss the internalization assay.

Reference

1. Haasen, D.; et al. G protein-coupled receptor internalization assays in the high-content screening format. Methods in enzymology. 2006, 414: 121-139.