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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 12 6107-6112
Copyright © 2003 by The Endocrine Society

COMMENT

Unexpected Effects of Epitope and Chimeric Tags on Gonadotropin-Releasing Hormone Receptors: Implications for Understanding the Molecular Etiology of Hypogonadotropic Hypogonadism

Shaun P. Brothers, Jo Ann Janovick and P. Michael Conn

Division of Neuroscience, Oregon National Primate Research Center and Departments of Physiology and Pharmacology and Cell and Developmental Biology, Oregon Health and Science University, Beaverton, Oregon 97006

Address all correspondence and requests for reprints to: P. Michael Conn, Oregon National Primate Research Center/Oregon Health and Science University, 505 NW 185th Avenue, Beaverton, Oregon 97006. E-mail: connm{at}ohsu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HYPOGONADOTROPIC HYPOGONADISM (HH) presents as a wide clinical spectrum, characterized by delayed sexual development and inappropriately low or apulsatile gonadotropin and sex steroid levels in the absence of anatomical or functional abnormalities of the hypothalamic-pituitary axis (1). This disorder is genetically heterogeneous and may be sporadic or familial (X-linked or autosomal). In patients without either anosmia or adrenal insufficiency, mutations in the gene encoding the GnRH receptor (GnRHR) are responsible for HH. This etiology was first reported in 1997 (2) as compound heterozygous mutations in a family with HH. Reports of inactivating mutations of the GnRHR are becoming more frequent; to date, 15 mutations of the GnRHR have been described (Fig. 1Go). Of these, one is a truncation mutant, nine are compound heterozygotes (2, 3, 4, 5, 6, 7, 8, 9, 10), and five are compound homozygotes (8, 11, 12). These mutations are widely distributed across the entire sequence of the GnRHR. Patients with GnRHR mutations exhibit a broad spectrum of phenotypes, ranging from partial to complete hypogonadism, even among affected kindred. Expression in heterologous cell systems that separately express each naturally occurring GnRHR mutant receptor show that some mutants are totally nonfunctional (E90K, A129D, R139H, S168R, A171T, C200Y, S217R, L266R, C279Y, and L314X), whereas others retain a modest degree of function (N10K, T32I, Q106R, R262Q, and Y284C) (Fig. 1Go). The commonly held belief that these mutations interfere with ligand binding or preclude interaction with effector proteins arose in part because of results obtained by hemagglutinin influenza virus (HA) epitope and green fluorescent protein (GFP) chimeric tagging, which suggested that such mutants were localized to the plasma membrane. The subsequent (and apparently contradictory) observation that pharmacological chaperones (pharmacoperones) (13) could rescue 12 of the 14 single-point mutation mutants suggested that protein misrouting was frequently the molecular etiology of normosmic, adrenal-sufficient HH. In the present study, we address this apparent contradiction in localization of the mutants and provide data suggesting that epitope and chimeric tagging alter cellular localization of the human GnRHR.



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  		FIG. 1. Sequence of the hGnRHR showing 15 known mutations isolated from patients with HH.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
General methods

Methods have been previously described for cDNA transfection, measurement of inositol phosphates (IP), and Scatchard analysis (14, 15). All mutants were transfected into COS7 cells as described (14). The identities of all cDNA coding sequences were reverified by Dye Terminator cycle sequencing, according to manufacturer’s instructions (Perkin-Elmer, Foster City, CA). After confirmation that 10-7 M buserelin was a saturating agonist concentration (data not shown), subsequent IP experiments used only this concentration of agonist. Data (n = 3) were analyzed using the paired t test (SigmaStat 3.0, Jandel Scientific Software, Chicago, IL; P < 0.05 was considered significant).

Literature evaluation

We examined the biomedical literature for the use of controls in studies using the HA-tag by using the National Library of Medicine Entrez/PubMED Web site (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi) with the search strategy:

((((((((hemagglutinin[Text Word] or ha[Text Word]) and (epitope[Text Word] or epitopes[Text Word])) and (tag[Text Word] or fusion protein[Text Word])) and English[Lang]) and ("1993"[PDat]: "3000"[PDat])) not hyaluronic acid[Text Word]) not histocompatibility antigen[Text Word]) not hydroxylamine[Text Word])

The results generated are publications written in English and published in the last 10 yr, with terms hemagglutinin or HA and epitope or epitopes and tag or fusion protein in text fields. Publications using the acronym HA for hyaluronic acid, histocompatibility antigen, or hydroxylamine were excluded by adding limiting search terms, as noted. This search resulted in 106 publications. Of these, we manually excluded 24 publications because either HA-tagging was described as part of a methodological paper with no experimental determinations (16 publications) or the full-length hemagglutinin protein was itself the focus of the research study (eight publications). Of the remaining 82 publications, we determined whether evaluative comparisons (such as ligand binding, effector coupling, protein localization, or other direct comparative assessments) of the HA-tagged and unmodified proteins were included as controls. Results tabulated for each year included in the literature evaluation and weighted data (based on the number, N, of publications for each year) was graphed and analyzed using SigmaPlot and SigmaStat (Jandel Scientific Software).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Human GnRHR

The sequence of the human GnRHR (hGnRHR) is shown in Fig. 1Go; this figure also shows the wide distribution of amino acid mutations associated with HH. Many of the mutated amino acids alter the local charge and, by inference, the local shape of the mutant receptor.

We measured a significant increase in IP production in response to GnRH agonist in cells expressing the HA-tagged wild-type (WT) hGnRHR, compared with cells expressing the untagged protein (P < 0.01, Fig. 2Go). When a dysfunctional mutant of the hGnRHR, hGnRHR(E90K), was HA-tagged, there was no significant change in IP production compared with the untagged protein (Fig. 2Go).



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  		FIG. 2. IP production for several hGnRHR and rGnRHR mutants transfected into COS7 cells. With a saturating concentration of agonist, an increase in IP production is observed when the HA-tagged human WT receptor or the HA-tagged rat mutant Ser326Ala expressing cells is compared with untagged receptor expressing cells. A similar increase in IP production is seen with several chimeric additions to the GnRHR(E90K) mutant. Data are representative of at least three independent experiments. (a, P < 0.01; b, P > 0.05; c, P = 0.027).

 
Primate GnRHRs (328 amino acids) contain an extra amino acid, K191, compared with rodent GnRHRs (327 amino acids); K191, a charged amino acid, significantly inhibits the percentage of synthesized receptor expressed at the plasma membrane, and, when removed from the receptor (des191), results in a 7-fold increase in plasma membrane expression (16). When K191 was deleted from the E90K mutant receptor [hGnRHR(E90K/des191)] and expressed in COS7 cells, IP production in response to agonist was significantly increased 14-fold (P < 0.002), compared with cells expressing hGnRHR(E90K). An even larger increase (18-fold, P < 0.002) was measured when the African catfish carboxyl-terminal 51 amino acids (Ctail) were added to GnRHR(E90K) along with the GFP chimeric tag [hGnRHR(E90K)-Ctail-GFP]. The des191, Ctail, and GFP modifications were approximately additive; the mutant hGnRHR(E90K/des191)-Ctail-GFP has the greatest IP response when expressed in COS7 cells [approximately 28-fold increase, compared with hGnRHR(E90K); P < 0.001].

Scatchard analysis was used to measure the affinity (Kd) and average numbers of expressed receptors per cell (N/C). We determined that the increase in IP production when comparing cells expressing the HA-tagged human WT receptor was due to a 70% increase in ligand affinity and not an increase in plasma membrane expression (Fig. 3Go). Only nonspecific binding (NSB) was measured with cells expressing hGnRHR(E90K) or HA-hGnRHR(E90K) because the slopes of the lines were not significantly distinct from zero (P > 0.05). When we added the Ctail/GFP sequence to the hGnRHR(E90K), we were able to measure specific ligand binding; Scatchard analysis of cells expressing hGnRHR(E90K/des191), hGnRHR(E90K)-Ctail-GFP, hGnRHR(E90K/des191)-Ctail, or hGnRHR(E90K/des191)-Ctail-GFP showed Kd = 2.672, 0.668, 0.550, and 0.551 nM, respectively, and N/C were 1831, 2241, 2875, and 3218, respectively (Fig. 3Go).



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  		FIG. 3. Scatchard analysis of binding data. After transient transfection, cells were exposed to a range of [125I]buserelin and allowed to equilibrate at room temperature; then cells were washed twice in PBS and bound radioactivity was determined. Slope and maximal binding (Bmax) were used to calculate ligand affinity (Kd) and average number of receptors per cell (N/C), respectively (inset tables; NSB, nonspecific binding).

 
Rat GnRHR

Previous comparative studies between the human and rat GnRHRs revealed that the rat GnRHR (rGnRHR) had significantly greater plasma membrane expression than the hGnRHR (13). Accordingly, a much higher proportion of the synthesized rGnRHR appears at the plasma membrane than the hGnRHR (13). To verify that the effects of tagging the hGnRHR were not species specific, we used a rGnRHR mutant with attenuated receptor-mediated IP production in COS7 cells [rGnRHR(Ser326Ala)] (15). When we compared the rat HA-tagged Ser326Ala [HA-rGnRHR(Ser326Ala)] mutant receptor to the nontagged receptor, we found that the cells expressing the HA-tagged mutant receptor produced significantly more IP, compared with cells expressing the untagged receptor (Fig. 2Go; P < 0.01). Furthermore, Scatchard analysis revealed that the numbers of receptors for rGnRHR(Ser326Ala) were significantly lower than the rat receptor and rat receptor derivatives, including HA-rGnRHR(Ser326Ala) (Fig. 3Go).

Literature evaluation

Forty-nine of 82 publications (60%) using the HA-tag failed to compare the untagged and tagged proteins. When we tabulated and plotted the weighted values corresponding to the percentage of the publications that included a control for the tagged protein, we found a negative, and significantly nonzero (P < 0.001), slope, indicating that fewer and fewer controls are being included in more recent studies (r2 = 0.223, Fig. 4Go). With deletion of either or both of the two outlier points (i.e. years 1993 and 1999), the negative slope remained significantly nonzero (P < 0.001) and the r2 values were 0.185, 0.418, and 0.454 for statistics recalculated without the 1993 data, 1999 data, or without both 1993 and 1999 data points, respectively.



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  		FIG. 4. Percentage of publications in the literature evaluation (see Materials and Methods) controlled for the addition of the HA-tag within each year of publication. The weighted trend has a downward slope (r2 = 0.223, P < 0.001), indicating progressively fewer publications included controls for the untagged protein. Solid line indicates the trend and dashed lines indicate the 95% confidence intervals (numbers shown, N, indicate the number of publications examined for each year).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The view that hGnRHR mutants associated with HH were correctly routed to the plasma membrane obscured understanding of the molecular etiology of this disease because mutants had, erroneously, been shown to be routed to the plasma membrane using either epitope or chimeric tagging in heterologous cell expression systems; occasionally, both HA and GFP chimeric tags are used simultaneously (16). Until recently it was unclear how to justify this view with the emerging data showing that most hGnRHR mutants were actually misrouted proteins.

The present study provides evidence that the addition of the HA-tag to the WT hGnRHR or a mutant of the rGnRHR causes increased IP production because of increased affinity or plasma membrane expression of the receptors, respectively, and suggests that data generated by employing these tags may not accurately reflect the cellular localization of modified proteins. We show that there is no change in IP production between the HA-tagged WT rGnRHR, compared with the untagged protein. This is not surprising in light of studies showing that the rGnRHR is fully expressed at the plasma membrane, with little intracellular retention of the synthesized receptor, whereas the hGnRHR is only partially expressed; expression of the hGnRHR, but not the rGnRHR, can be increased by the pharmacoperone, IN3, and others (14). The Ser326Ala mutation causes about 45% of mutant rGnRHR to be retained in the cell, and adding the HA-tag to the mutant completely reverses this effect. The Ctail/GFP chimera of hGnRHR(E90K) also significantly enhances plasma membrane expression and results in an IP response to the GnRHR agonist of an otherwise nonfunctional HH mutant.

The single amino acid deletion of K191 that had been shown to increase plasma membrane expression of the WT hGnRHR (17) also has the same action when removed from the E90K HH mutant because cells expressing this mutant [hGnRHR(E90K/des191)] produced significantly more IP and expressed more receptors, compared with cells expressing hGnRHR(E90K). Similarly, when the Ctail-GFP sequence was added to the hGnRHR(E90K) [hGnRHR(E90K)-Ctail-GFP], we measured increased plasma membrane expression and IP production, compared with cells expressing hGnRHR(E90K). An approximately additive increase in IP production was measured when a mutant construct bearing all the expression-increasing mutations was stimulated with agonist, hGnRHR(E90K/des191)-Ctail-GFP, when expressed in COS7 cells produced 28-fold more IP, compared with cells expressing the hGnRHR(E90K); although no change in ligand affinity was observed, a 12% increase in plasma membrane expression was also observed, compared with cells expressing the untagged hGnRHR(E90K/des191)-Ctail, entirely because of the presence of the GFP, a protein larger than the entire hGnRHR molecule. We do not suggest that all the modifications reported here necessarily alter the conformation of the GnRHR or that they can completely rescue a protein. In the case of the GFP, for example, because this protein is larger than the GnRHR, it is likely that the GFP may be a significant determinant of the routing of the chimera. The lack of predictability of the effect of GFP or epitope tags on a protein strengthens the case for the use of controls for such experiments.

When we evaluated the literature, we were surprised to find that the majority of publications using the HA-tag failed to include any data comparing the tagged and untagged proteins; although we did not evaluate GFP chimeric tagging studies, we would expect to find a similar pattern. We also found that publications reporting controls comparing WT and HA-tagged proteins are declining, a particularly troubling trend, likely reflecting greater editorial acceptance of these approaches.

The literature contains data suggesting other surprising differences between tagged and WT proteins, as well. For instance, Tolbert and Lameh (18) found that the human M1-muscarinic receptor, epitope tagged at the amino terminus, internalized with the addition of the antiepitope antibody even in the absence of other ligands. Similarly, Ledent et al. (19) found that the addition of six histidine residues (6xHis-tag) to the carboxyl terminus of ß-lactamase resulted in an amino-terminal truncation of the protein by disrupting the signal peptidase site of action. Furthermore, Romano et al. (20) found that the addition of the HA-tag to the amino terminus or carboxy terminus (or to an internal site) disrupts the endoplasmic reticulum localization of Ste14p and resulted in unexpected targeting to the Golgi.

Pharmacological rescue of mutant GnRH receptors associated with HH by peptidomimetics increases plasma membrane expression of at least 13 HH mutations and human, but not rodent, WT GnRHR [Refs. 21 and 22 , and unpublished data showing pharmacological rescue of the GnRHR(A171T) HH mutant receptor]. This latter observation emphasizes the apparent role of partial plasma membrane expression of synthesized GnRHR, an event that appears largely associated with primates. It is possible that this level of regulation is generally more important for G protein-coupled receptors than has been previously appreciated. Experiments by Krautwurst et al. (23) showed that chimeric addition of the amino-terminal 20 amino acids from the rhodopsin G protein-coupled receptor to members of the (difficult to express) odorant receptor family, enhanced plasma membrane expression. Similarly, Koller et al. (24) found about 50% more FSH receptors were expressed on the plasma membrane after adding the HA-tag. FSH receptor expression is tightly regulated over the course of the menstrual cycle (25). Furthermore, a view has been presented that the gene product that gives rise to the human {delta}-opioid receptor is inefficiently processed, with only about half of the translation product reaching the membrane (26).

Membrane surface expression patterns can be regulated by natural ligands and homeostatic posttranslational modifications; similarly, we, and others, have shown that specific pharmacological chaperones can rescue plasma membrane expression of the GnRH and vasopressin V2 receptors as well as rescue misfolded mutant cytoplasmic proteins: lysosomal {alpha}-galactosidase A and p53 tumor suppressor mutants (17, 21, 27, 28, 29). Other chaperones, including polyols, dimethylsulfoxide, and trimethyl amines are also known to stabilize the native structure of many proteins offering a limited, nonspecific means of rescue (30). These reports cause reevaluation of the molecular etiology of misfolding diseases (21, 31, 32) and present new therapeutic options (27).

In vitro, site-directed mutagenesis of the human GnRHR has shown that the deletion of the positively charged residue K191 increases plasma membrane receptor expression, as does the chimeric addition of the African catfish GnRHR carboxyl-terminal 51 amino acids (Ctail; sequence, TPSFRADLSRCFCWRNQNASAKSLPHFSGHRREVSGEAESDLGSGDQPS-GQ) to mammalian GnRHRs (17, 33, 34). Alternatively, other mutations that abolish GnRHR function are widely distributed in the GnRHR and often involve charge or polarity changes in amino acid composition (Fig. 1Go). The net negatively charged HA-tag sequence (Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) from the hemagglutinin influenza virus has proven useful in a wide variety of proteomic applications (35, 36, 37, 38, 39, 40). Comparisons of original and HA-tagged versions of proteins are relatively infrequent in the literature, however. Often, proteins are tagged with the assumption that the modification does not affect change in protein surface expression or function, an erroneous assumption in the example of HH GnRHRs. Impaired intracellular routing of mutant human GnRHRs has gone unnoticed in previous studies as a mechanism adding to the loss of function of HH receptors because of hindrance by epitope and chimeric tags. We propose that controls should be used to directly compare the tagged with the unmodified protein, an idea not regularly implemented to date. Epitope and chimeric tags provide potentially valuable research tools when used sensibly; we and others (20) suggest caution should be used when interpreting experimental outcomes. A side-by-side comparison of physical characteristics including cellular localization, ligand binding, and effector coupling between epitope or chimeric tagged and unmodified proteins should be viewed as an essential part of any study in which these tags are used.


   Acknowledgments
 
We thank Oregon National Primate Research Center librarians Cooky Abrams and Denise Urbanski for obtaining copies of all 106 publications in our literature search.


   Footnotes
 
This work was supported by National Institutes of Health Grants HD-19899, RR-00163, and HD-18185.

Abbreviations: Ctail, Terminal 51 amino acid cytoplasmic carboxyl terminus of the African catfish GnRHR; GFP, green fluorescent protein; GnRHR, GnRH receptor; HA, hemagglutinin influenza virus epitope; HH, hypogonadotropic hypogonadism; IP, inositol phosphates; N/C, average numbers of expressed receptors per cell; WT, wild type.

Received June 18, 2003.

Accepted September 2, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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