Abstract
Epitopes accessible on the surface of intact cells are extremely valuable in studies of membrane proteins, allowing quantification and determination of the distribution of proteins as well as identification of cells expressing large numbers of proteins. However for many membrane proteins there are no suitable antibodies to native sequences, due to lack of availability, low affinity or lack of specificity. In these cases the use of an introduced epitope at specific sites in the protein of interest can often provide a suitable tool for studies. However, the introduction of the epitope sequence has the potential to affect protein expression, the assembly of multisubunit proteins or transport to the surface membrane. We find that surface expression of heteromeric neuronal nicotinic receptors containing the α4 and β4 subunits can be affected by introduced epitopes when inserted near the amino terminus of a subunit. The FLAG epitope greatly reduces surface expression when introduced into either α4 or β4 subunits, the V5 epitope has little effect when placed in either, while the Myc epitope reduces expression more when inserted into β4 than α4. These results indicate that the extreme amino terminal region is important for assembly of these receptors, and demonstrate that some widely used introduced epitopes may severely reduce surface expression.
Highlights
Receptors for neurotransmitters mediate cellular responses to extracellular ligands and their known physiological role requires that they be expressed on the surface membrane of cells, often in particular regions
We and others have inserted the FLAG epitope (DYKDDDDK) near the amino-terminal end of GABAA receptor subunits without reducing surface expression [3, 4]
We used a monoclonal antibody to the α4 subunit that recognizes an extracellular epitope to examine the level of surface expression in enzyme-linked immunosorbent assays (ELISA) assays on intact cells
Summary
Receptors for neurotransmitters mediate cellular responses to extracellular ligands and their known physiological role requires that they be expressed on the surface membrane of cells, often in particular regions (e.g. subsynaptic membrane). For this reason it is valuable to have probes for the presence of these receptors that recognize them in intact cells, in normal conditions. Some receptors have small molecule or toxin probes that associate with extracellular regions and can be used for this purpose, but antibodies to either native or introduced epitopes in the extracellular domain are the most widely used reagents. The use of introduced sequences raises the possibility that expression of the mature receptor on the PLOS ONE | DOI:10.1371/journal.pone.0151071 March 10, 2016
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