Ideal Methods to Study Cellular Transport of Membrane Proteins
Translocation and Trafficking of Membrane Proteins
Membrane proteins (such as receptors like GPCRs and ion channels) are translated and undergo proper folding in the endoplasmic reticulum (ER) and then transported to the cell surface (plasma membrane). Additionally, as part of the normal recycling and receptor activation processes, membrane proteins internalize from the plasma membrane into endosomes. The appropriate localization of membrane proteins is essential to maintain their biological function, but in some cases, these protein transport events are altered leading to a non-functional membrane protein and undesirable effects that are often associated with serious human diseases (e.g. cystic fibrosis, Alzheimer’s disease, and Huntington’s disease).
Explore Eurofins DiscoverX products and solutions related to these methods: Translocation assays, GPCR Internalization assays, and Pharmacotrafficking assays.
Preventing Undesirable Translocation Events
Membrane protein defects, caused by mutations, deletions, or truncations, can lead to protein misfolding in the ER and prevent their trafficking to the plasma membrane. This mislocalization renders the membrane protein non-functional and leads to diseases like cystic fibrosis. Many endogenous or synthetic receptor ligands (like morphine) can change the rate of membrane protein internalization or recycling. Drug-induced receptor internalization or recycling is an important pharmacological property since it can limit their clinical effectiveness and result in unwanted side effects, tachyphylaxis, or drug tolerance problems.
The detrimental cellular events caused by protein mislocalization and excessive protein internalization can be reversed by the development of compounds called pharmacochaperones (short for pharmacological chaperones) and better drugs (like partial agonists), respectively. Pharmacochaperones correct the folding of the abnormally misfolded proteins allowing them to translocate properly from the ER to the plasma membrane. Once at the cell surface, the partial agonist can bind to the membrane protein initiating a partially efficacious response compared to a full agonist, while also reducing unwanted side effects and preventing other internalization issues.
Methods of Measuring Subcellular Translocation of Membrane Proteins
Researchers currently monitor membrane protein translocation events using the high content imaging methods outlined below.
- Immunohistochemistry (IHC) or immunocytochemistry (ICC) – Provides visualization of membrane protein localization
- Fluorescence-activated cell sorting (FACS) – Provides quantitative detection of membrane protein localization
Problems with high content imaging methods:
- Low throughput
- Require antibodies or fluorescent tags
- Use specialized equipment that can often be expensive
In contrast, cell-based assays for studying protein function, trafficking, and internalization are becoming more mainstream for screening and profiling potential drugs to prevent translocation issues.
Benefits of using cell-based assays for studying translocation issues:
- High throughput
- Quantitative
- Affordable with no need for specialized equipment, assays can be read using a standard luminometer
These methods are complementary to each other and should all be considered when studying cellular membrane protein translocation events.
Membrane Protein Translocation Resources
Learn how Eurofins DiscoverX can help your research into protein translocation events through the use of our easy-to-use, quantitative, and high throughput cell-based assays.
- Translocation assays to monitor trafficking of proteins to the plasma membrane, endosomes and nucleus
- Pharmacotrafficking assays to discover novel pharmacochaperones for abnormal GPCRs and ion channels
- Parental cell lines to generate your own translocation assay
- Read an application note: Quantify GPCR Endocytosis and Recycling with PathHunter GPCR Internalization Assays – Analyzing Therapeutics for Opioid and Cholecystokinin Receptors
- Listen to webinars
