Loading...

Blog Article

The Trouble with Chemokine Receptors

Chemokine receptors are G protein-coupled receptors that allow cells to migrate towards increasing chemokine gradients. They are vital in many processes of immune cells during their lifetime, including homeostasis and inflammation.

Chemokine receptors are divided into different families based on the type of chemokine that they bind: CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors and XC chemokine receptors.
Structure

What makes the chemokine receptor unique is its three dimensional structure in the context of the cell membrane. While most cell surface molecules are type I or type II single pass proteins, the chemokine receptors pass through the membrane seven times. This has several implications for the molecule:
  1. It is more likely to be affected by the nearby lipids in the membrane, such as cholesterol, and gangliosides, in addition to phospholipids.
  2. Instead of having rigid globular structures like other proteins, it has 3 loops and an N-terminus structure on the external side of the cell membrane. Because these loops lack beta-barrels or alpha helixes, their structure is not as rigid as folded globular proteins.

A slinky is probably more rigid than a chemokine receptor in the membrane.
  1. The receptor is designed to change shape upon binding to its ligand, in order to transmit a signal to the interior of the cell. This means that there must be a reorganization of the seven transmembrane domains, again suggesting the lack of rigidity of the molecule in the context of the membrane.
  2. Lastly, extensive post-translational modifications could also affect the structure, particularly large O-linked glycosylation sites externally, as well as palmitoylation and phosphorylation sites on the intracellular side.
So why is this troublesome?

Antibodies.

The successful production of chemokine receptor specific antibodies can be quite difficult, particularly if you are trying to develop an antibody for flow cytometry, which needs to recognize the protein in its native state in the membrane. The reasons mentioned above regarding the structure are likely reasons why it would be difficult. Furthermore, once you have generated an antibody, it can still be difficult to detect the receptor. The structure, or flexibility of the structure, may be the real culprit.
Imagine that you are an antibody looking for its chemokine receptor target on the cell surface. From the top view you might bind to an epitope that spans multiple loops like this (circled in red):







If the positioning of those loops changes, even slightly, due to membrane lipid changes, temperature changes, glycosylation changes, or other influencing factors, one could easily see how the antibody might lose its binding ability:
What can you do about it?

Whether you are immunophenotyping, or blocking the receptor, you will need to start with good antibodies that are specific and have high affinity. BioLegend provides an excellent selection of anti-chemokine receptor antibodies, covering a majority of all receptors for mouse and humans. To give yourself confidence, verify them against other antibodies you may be currently using. There are also protocol techniques that can help you optimize staining:
  • Try staining at 37°C or room temperature. This depends on the antibody; some work better at lower temperatures, but most work best at room temperature or 37°C.
  • If receptor expression is low, use a bright fluorophore conjugate such as Brilliant Violet 421™ or PE to get maximal signal-to-noise.
  • Avoid using sodium azide in the staining solution as this is known to decrease binding for some antibodies.
  • Verify the protocol used by the antibody manufacturer for quality control, since the cell preparation may affect antibody binding to the chemokine receptor: whole blood, lysed whole blood, FICOLL-prepared PBMCs, fixed cells, etc.
Learn more about Chemokines and Chemokine Receptors on our webpage.
The following papers are relevant, useful reading:

Contributed by Dzung Nguyen, PhD.
Related Pages
ProductsHere
Login / Register
Remember me
Forgot your password? Reset password?
Create an Account