title: Introduced Amino Terminal Epitopes Can Reduce Surface Expression of Neuronal Nicotinic Receptors.
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.
authors.affiliation: Department of Anesthesiology, Washington University School of Medicine, Saint Louis, MO, United States of America.
authors.forename: John R
authors.initials: JR
authors.lastname: Bracamontes
authors.affiliation: Department of Anesthesiology, Washington University School of Medicine, Saint Louis, MO, United States of America.|Taylor Family Institute for Innovative Psychiatric Research, Washington University School of Medicine, Saint Louis, MO, United States of America.
authors.forename: Gustav
authors.initials: G
authors.lastname: Akk
authors.affiliation: Department of Anesthesiology, Washington University School of Medicine, Saint Louis, MO, United States of America.|Taylor Family Institute for Innovative Psychiatric Research, Washington University School of Medicine, Saint Louis, MO, United States of America.
authors.forename: Joe Henry
authors.initials: JH
authors.lastname: Steinbach
mesh_terms: D000939:Epitopes / Q000096:biosynthesis / Q000235:genetics; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D011978:Receptors, Nicotinic / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000939:Epitopes; D011978:Receptors, Nicotinic