Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Am J Physiol Cell Physiol
2009 Sep 01;2973:C591-600. doi: 10.1152/ajpcell.00166.2009.
Show Gene links
Show Anatomy links
Agonist-induced internalization and downregulation of gonadotropin-releasing hormone receptors.
Finch AR
,
Caunt CJ
,
Armstrong SP
,
McArdle CA
.
???displayArticle.abstract???
Gonadotropin-releasing hormone (GnRH) acts via seven transmembrane receptors to stimulate gonadotropin secretion. Sustained stimulation desensitizes GnRH receptor (GnRHR)-mediated gonadotropin secretion, and this underlies agonist use in hormone-dependent cancers. Since type I mammalian GnRHR do not desensitize, agonist-induced internalization and downregulation may underlie desensitization of GnRH-stimulated gonadotropin secretion; however, research focus has recently shifted to anterograde trafficking, with the finding that human (h)GnRHR are mostly intracellular. Moreover, there is little direct evidence for agonist-induced trafficking of hGnRHR, and whether or not type I mammalian GnRHR show agonist-induced internalization is controversial. Here we use automated imaging to monitor expression and internalization of hemagglutinin (HA)-tagged hGnRHRs, mouse (m) GnRHR, Xenopus (X) GnRHRs, and chimeric receptors (hGnRHR with added XGnRHR COOH tails, h.XGnRHR) expressed by adenoviral transduction in HeLa cells. We find that agonists stimulate downregulation and/or internalization of mGnRHR and XGnRHR, that GnRH stimulates trafficking of hGnRHR and can stimulate internalization or downregulation of hGnRHR when steps are taken to increase cell surface expression (addition of the XGnRHR COOH tail or pretreatment with pharmacological chaperone). Agonist effects on internalization (of h.XGnRHR) and downregulation (of hGnRHR and h.XGnRHR) were not mimicked by a peptide antagonist and were prevented by a mutation that prevents GnRHR signaling, demonstrating dependence on receptor signaling as well as agonist occupancy. Thus agonist-induced internalization and downregulation of type I mammalian GnRHR occurs in HeLa cells, and we suggest that the high throughput imaging systems described here will facilitate study of the molecular mechanisms involved.
Fig. 1. Quantification of hemagglutinin (HA)-tagged gonadotropin-releasing hormone (GnRH) receptors in HeLa cells by fluorescence immunohistochemistry with semiquantitative image acquisition and analysis. HeLa cells were transduced with the adenovirus (Ad)-expressing HA-tagged hGnRHRs, human Xenopus GnRHRs (h.XGnRHRs), or XGnRHRs [1 plaque-forming unit (pfu)/nl] and then incubated 18 h in medium with 0 or 10−7 M IN3. They were then stained for cell surface receptors [incubation of intact cells with primary antibody (Ab)], and nuclei were stained with 4′,6-diamidino-2-phenyindole (DAPI), before image acquisition and analysis as described in materials and methods. To measure whole cell receptor expression, permeabilized cells were exposed to primary and secondary Ab before imaging as above. A: thumbnail images from individual wells stained for cell surface and whole cell receptor expression after transduction with Ad GnRHR. CTRL, control. B: higher magnification for a region of cells (IN3-treated cell surface HA-hGnRHRs from the white-boxed thumbnail region in A) stained for nuclei (DAPI, blue) or GnRHR (green). The merged image illustrates the perimeters of the nuclei and cells determined using IN Cell 1000 Analyzer software and the application of a filter to define positively (+ve) stained cells (HA-hGnRHR staining >20% above background - green perimeter traces) and negatively stained (−ve) cells (HA-hGnRHR staining <20% above background - red perimeter traces). C: expression index (EI) calculated by compounding the percentage of +ve cells by the mean receptor fluorescence intensity (arbitrary units). D: proportional cell surface expression (PCSE) calculated by expressing the cell surface EI as a percentage of the whole cell EI. These data are all from the same representative experiment, and bar charts show means ± SE for 4–8 replicate wells (i.e., data derived from >5,000 individual cells).
Fig. 2. Agonist and antagonist effects on GnRHR expression at the cell surface. A and B: HeLa cells transduced with HA-hGnRHR or HA-h.XGnRHR were treated as described for Fig. 1 except that they were incubated for 18 h with 10−7 M IN3, 10−7 M Buserelin (Bus), or with no addition (CTRL) before staining for HA-tagged GnRHRs in intact cells (cell surface) and permeabilized cells (whole cell) as above. Data shown are the surface expression index (arbitrary units) and PCSE determined as described above. C–F: HeLa cells transduced with HA-hGnRHR, HA-h.XGnRHR, HA-mouse (m) GnRHR, or HA XGnRHR were incubated for 18 h with 10−7 M IN3 (●) or with no addition (○) before being washed and incubated for the indicated periods with 10−7 M GnRH (C–E) or GnRH II (F) at 37°C. These incubations were terminated by washing at 4°C, before staining for cell surface HA-tagged GnRHRs. Data shown are the surface expression index (arbitrary units) determined as above and normalized to the internal control (IN3 treated for C–E). All values are means ± SE (n = 3) from 3 separate experiments each with 3–6 replicate wells. In F, the data obtained with and without IN3 pretreatment were indistinguishable and have therefore been pooled. *P < 0.05, **P < 0.01 compared with CTRL.
Fig. 3. Dose dependence of agonist effects on cell surface GnRHR expression. HeLa cells transduced with HA-hGnRHR or HA-h.XGnRHR (1 pfu/ml) were incubated for 18 h with 10−7 M IN3 (●) or with no addition (○) before being washed and incubated for 2 h with the indicated concentration of Buserelin at 37°C. These incubations were terminated by washing at 4°C, before staining for cell surface HA-tagged GnRHRs. Data shown are the surface expression index (arbitrary units) determined and normalized as above and are means ± SE (n = 3) from 3 separate experiments each with 3 replicate wells.
Fig. 4. Signal dependence of agonist effects on cell surface GnRHR expression. HeLa cells transduced with HA-hGnRHR or HA-h.XGnRHR with or without the A261K mutation (1 pfu/nl) were incubated for 18 h with 10−7 M IN3 (●) or with no addition (○) before being washed and incubated for the indicated periods with 10−7 M Buserelin at 37°C. These incubations were terminated by washing at 4°C, before staining for cell surface HA-tagged GnRHRs. Data shown are the surface expression index (arbitrary units) determined as above and are means ± SE (n = 3) from 3 separate experiments (each with 3 replicate wells per treatment) and are normalized to the internal control value. **P < 0.01 compared with CTRL.
Fig. 5. Anti-HA loading: time and agonist dependence. Top rows: representative images from HeLa cells transduced with h.XGnRHR (no HA tag) or from cells expressing HA-tagged h.XGnRHR before being washed and incubated 30 min at 37°C with primary Ab (mouse anti-HA at 1:200) with 0 or 10−7 M GnRH as shown. These incubations were terminated by washing at 4°C, before fixation, permeabilization, and staining (DAPI and Alexa Fluor 488-conjugated anti-mouse IgG), followed by image acquisition and analysis. Bottom row: illustration of the use of automated algorithms to segment the images, defining the perimeters of the nuclei (from the DAPI stain), adding a 2-μm collar, and identifying the bright punctuate regions of HA-GnRHR stain within the cross-sectional area defined by nucleus and collar. This number (“inclusion count,” the mean number of the small circles in the segmented images) provides a measure of receptors that have trafficked from the plasma membrane to putative endosomes. In the bottom panels, HeLa cells transduced with HA-tagged hGnRHR, h.XGnRHR, A261K-h.XGnRHR, or XGnRHR (1 pfu/nl) were incubated for 0–60 min at 37°C in medium with primary antibody and 0 (○) or 10−7 M GnRH (●, GnRH II for the XGnRHR) as indicated. Nontagged versions of three of the GnRHRs were also included as negative controls (▵). The cells were washed, fixed, permeabilized, and stained before image acquisition and analysis, as above. The data shown are inclusion counts as means ± SE (n = 3) pooled from 4 separate experiments (each with 4 replicate wells per treatment). *P < 0.05, **P < 0.01 compared with corresponding control value without GnRH.
Fig. 6. Agonist-induced redistribution of HA-GnRHR. A and B: HeLa cells transduced with HA-tagged hGnRHR, h.XGnRHR, or mGnRHR or with nontagged h.XGnRHR as a negative control (HA-h, HA-h.X, HA-m, and h.X, respectively) were washed and incubated for 60 min at room temperature (∼21°C) in medium with anti-HA (1:200). They were then washed and incubated for 15 min (A) or 60 min (B) at 37°C in medium with 0 or 10−7 M GnRH as indicated before being processed for determination of inclusion count, as above. GnRH effects were only statistically significant (**P < 0.01 compared with corresponding control) after 60 min in cells expressing HA-h.XGnRHR or HA-mGnRHR. C: HeLa cells transduced with HA-h.XGnRHR (●) or HA-hGnRHR (○) were loaded with primary Ab as above, then washed and incubated for 60 min at 37°C in medium with the indicated concentration of GnRH. D–F: HeLa cells transduced with HA-h.XGnRHR, HA-mGnRHR, or HA-A261K h.XGnRHR were prepared and loaded with anti-HA as above, before being washed and incubated for 60 min at 37°C in medium with no addition (CTRL), GnRH (10−9 M), cetrorelix (Cetro; 10−7 M), IN3 (10−7 M), or with GnRH plus cetrorelix (G + C) or GnRH plus IN3 (G + I) as indicated. In all cases, incubations were terminated by washing at 4°C and cells were then processed to determine the inclusion count, as described in materials and methods. The data shown are means ± SE (n = 3 or 4) pooled from 3 or 4 separate experiments (each with 2–4 replicate wells per treatment). ANOVAs revealed “treatment” as a significant variable in A–E (but not in F). NS, not significant. **P < 0.01 compared with corresponding control without GnRH.
Bernier,
Pharmacological chaperones: potential treatment for conformational diseases.
2004, Pubmed
Bernier,
Pharmacological chaperones: potential treatment for conformational diseases.
2004,
Pubmed
Blomenröhr,
Pivotal role for the cytoplasmic carboxyl-terminal tail of a nonmammalian gonadotropin-releasing hormone receptor in cell surface expression, ligand binding, and receptor phosphorylation and internalization.
1999,
Pubmed
Caunt,
Regulation of gonadotropin-releasing hormone receptors by protein kinase C: inside out signalling and evidence for multiple active conformations.
2004,
Pubmed
,
Xenbase
Caunt,
Arrestin-mediated ERK activation by gonadotropin-releasing hormone receptors: receptor-specific activation mechanisms and compartmentalization.
2006,
Pubmed
,
Xenbase
Cheng,
Molecular biology of gonadotropin-releasing hormone (GnRH)-I, GnRH-II, and their receptors in humans.
2005,
Pubmed
Conn,
Potency enhancement of a GnRH agonist: GnRH-receptor microaggregation stimulates gonadotropin release.
1982,
Pubmed
Conn,
The molecular mechanism of action of gonadotropin releasing hormone (GnRH) in the pituitary.
1987,
Pubmed
Conn,
Protein folding as posttranslational regulation: evolution of a mechanism for controlled plasma membrane expression of a G protein-coupled receptor.
2006,
Pubmed
Conn,
G protein-coupled receptor trafficking in health and disease: lessons learned to prepare for therapeutic mutant rescue in vivo.
2007,
Pubmed
Cooray,
Accessory proteins are vital for the functional expression of certain G protein-coupled receptors.
2009,
Pubmed
Cornea,
Simultaneous and independent visualization of the gonadotropin-releasing hormone receptor and its ligand: evidence for independent processing and recycling in living cells.
1999,
Pubmed
Davidson,
Absence of rapid desensitization of the mouse gonadotropin-releasing hormone receptor.
1994,
Pubmed
Davidson,
Identification of N-glycosylation sites in the gonadotropin-releasing hormone receptor: role in receptor expression but not ligand binding.
1995,
Pubmed
Dong,
Regulation of G protein-coupled receptor export trafficking.
2007,
Pubmed
Edwards,
Localization of G-protein-coupled receptors in health and disease.
2000,
Pubmed
Ellgaard,
Setting the standards: quality control in the secretory pathway.
1999,
Pubmed
Finch,
Plasma membrane expression of GnRH receptors: regulation by antagonists in breast, prostate, and gonadotrope cell lines.
2008,
Pubmed
,
Xenbase
Hanyaloglu,
Regulation of GPCRs by endocytic membrane trafficking and its potential implications.
2008,
Pubmed
Hazum,
Receptor-mediated internalization of fluorescent gonadotropin-releasing hormone by pituitary gonadotropes.
1980,
Pubmed
Heding,
Gonadotropin-releasing hormone receptors with intracellular carboxyl-terminal tails undergo acute desensitization of total inositol phosphate production and exhibit accelerated internalization kinetics.
1998,
Pubmed
Hislop,
Desensitization and internalization of human and xenopus gonadotropin-releasing hormone receptors expressed in alphaT4 pituitary cells using recombinant adenovirus.
2000,
Pubmed
,
Xenbase
Hislop,
Differential internalization of mammalian and non-mammalian gonadotropin-releasing hormone receptors. Uncoupling of dynamin-dependent internalization from mitogen-activated protein kinase signaling.
2001,
Pubmed
,
Xenbase
Janovick,
Structure-activity relations of successful pharmacologic chaperones for rescue of naturally occurring and manufactured mutants of the gonadotropin-releasing hormone receptor.
2003,
Pubmed
Jennes,
Receptor-mediated uptake of GnRH agonist and antagonists by cultured gonadotropes: evidence for differential intracellular routing.
1986,
Pubmed
Lin,
Addition of catfish gonadotropin-releasing hormone (GnRH) receptor intracellular carboxyl-terminal tail to rat GnRH receptor alters receptor expression and regulation.
1998,
Pubmed
Luttrell,
The role of beta-arrestins in the termination and transduction of G-protein-coupled receptor signals.
2002,
Pubmed
McArdle,
Signalling, cycling and desensitisation of gonadotrophin-releasing hormone receptors.
2002,
Pubmed
,
Xenbase
McArdle,
Desensitization of gonadotropin-releasing hormone action in the gonadotrope-derived alpha T3-1 cell line.
1995,
Pubmed
McArdle,
Desensitization of gonadotropin-releasing hormone action in alphaT3-1 cells due to uncoupling of inositol 1,4,5-trisphosphate generation and Ca2+ mobilization.
1996,
Pubmed
Millar,
Gonadotropin-releasing hormone receptors.
2004,
Pubmed
Myburgh,
Alanine-261 in intracellular loop III of the human gonadotropin-releasing hormone receptor is crucial for G-protein coupling and receptor internalization.
1998,
Pubmed
Pawson,
Contrasting internalization kinetics of human and chicken gonadotropin-releasing hormone receptors mediated by C-terminal tail.
1998,
Pubmed
Pawson,
Mammalian type I gonadotropin-releasing hormone receptors undergo slow, constitutive, agonist-independent internalization.
2008,
Pubmed
Petaja-Repo,
Export from the endoplasmic reticulum represents the limiting step in the maturation and cell surface expression of the human delta opioid receptor.
2000,
Pubmed
Petaja-Repo,
Newly synthesized human delta opioid receptors retained in the endoplasmic reticulum are retrotranslocated to the cytosol, deglycosylated, ubiquitinated, and degraded by the proteasome.
2001,
Pubmed
Pierce,
Seven-transmembrane receptors.
2002,
Pubmed
Schvartz,
Internalization and recycling of receptor-bound gonadotropin-releasing hormone agonist in pituitary gonadotropes.
1987,
Pubmed
Sedgley,
Intracellular gonadotropin-releasing hormone receptors in breast cancer and gonadotrope lineage cells.
2006,
Pubmed
,
Xenbase
Stojilkovic,
Expression and signal transduction pathways of gonadotropin-releasing hormone receptors.
1995,
Pubmed
Tan,
Membrane trafficking of G protein-coupled receptors.
2004,
Pubmed
Ulloa-Aguirre,
Pharmacologic rescue of conformationally-defective proteins: implications for the treatment of human disease.
2004,
Pubmed
Vrecl,
Agonist-induced endocytosis and recycling of the gonadotropin-releasing hormone receptor: effect of beta-arrestin on internalization kinetics.
1998,
Pubmed
Willars,
Rapid down-regulation of the type I inositol 1,4,5-trisphosphate receptor and desensitization of gonadotropin-releasing hormone-mediated Ca2+ responses in alpha T3-1 gonadotropes.
2001,
Pubmed
Zhang,
Role of delivery and trafficking of delta-opioid peptide receptors in opioid analgesia and tolerance.
2006,
Pubmed