Unexpected effects of epitope and chimeric tags on gonadotropin-releasing hormone receptors: implications for understanding the molecular etiology of hypogonadotropic hypogonadism.
In the case of human GnRH receptor (GnRHR) mutants associated with hypogonadotropic hypogonadism, a view emerged that these mutants are correctly routed to the plasma membrane. This view, supported almost entirely by studies using the HA-tag (hemagglutinin influenza virus epitope tag) and other epitope and chimeric tags, obscured recognition that GnRHR mutants frequently become misrouted proteins. The underlying assumption in epitope and chimeric tagging studies is that the cell does not distinguish tagged from unmodified proteins. It should not have been surprising, in retrospect, to find that even a single amino acid mutation dramatically alters protein function or routing because increased plasma membrane expression is associated with deletion of a single amino acid in the human GnRHR (K191), and point mutations have been shown to block plasma membrane routing of many receptors, including most of those responsible for the hypogonadotropic hypogonadism phenotype. Our present observations suggest that epitope and chimeric tags do have a significant effect on protein localization and function. Although rarely provided, control experiments addressing the effects of epitope or chimeric tagging are an essential part of any study relying on these proteomic tools.
Brothers
Shaun P
SP
Oregon National Primate Research Center and Department of Physiology, Oregon Health and Science University, Beaverton, Oregon 97006, USA.
Janovick
Jo Ann
JA
Conn
P Michael
PM
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D019556:COS Cells; D000939:Epitopes; D006801:Humans; D007006:Hypogonadism / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D051381:Rats; D011966:Receptors, LHRH / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D016324:Sequence Tagged Sites; D014018:Tissue Distribution
D000939:Epitopes; D011966:Receptors, LHRH; D011993:Recombinant Fusion Proteins