Using automated imaging to interrogate gonadotrophin-releasing hormone receptor trafficking and function

Abstract

Gonadotrophin-releasing hormone (GnRH) acts via seven transmembrane receptors on gonadotrophs to stimulate gonadotrophin synthesis and secretion, and thereby mediates central control of reproduction. Type I mammalian GnRHR are unique, in that they lack C-terminal tails. This is thought to underlie their resistance to rapid homologous desensitisation as well as their slow rate of internalisation and inability to provoke G-protein-independent (arrestin-mediated) signalling. More recently it has been discovered that the vast majority of human GnRHR are actually intracellular, in spite of the fact that they are activated at the cell surface by a membrane impermeant peptide hormone. This apparently reflects inefficient exit from the endoplasmic reticulum and again, the absence of the C-tail likely contributes to their intracellular localisation. This review is intended to cover some of these novel aspects of GnRHR biology, focusing on ways that we have used automated fluorescence microscopy (high content imaging) to explore GnRHR localisation and trafficking as well as spatial and temporal aspects of GnRH signalling via the Ca(2+)/calmodulin/calcineurin/NFAT and Raf/MEK/ERK pathways.

Fig. 1

An automated imaging assay for GnRHR quantification. Cells grown in 96 wells were transduced with Ad expressing N-terminal HA-tagged hGnRHR, XGnRHR or h.XGnRHR then incubated ∼20 h with 0 or 1 μM of the non-peptide antagonist IN3 before indirect fluorescence labelling of cell surface receptors (primary antibody added to intact cells) or whole cell receptors (primary antibody added after permeabilisation). Nuclei were also stained with DAPI and digital images were captured using a 10× objective and a 0.6 mm² field of view. Panel A shows representative images (each approximately 25% of the field captured) of whole cells and cell surface staining in cells transduced with the indicated receptors. Panel B shows a higher power image of nuclei, HA-XGnRHR and merged stains from the boxed region in panel A. It also illustrates the automated image segmentation used to define perimeters of nuclei (blue) and cells (green or red) and application of a filter to distinguish cells in which staining was >10% above background (green perimeters) or <10% above background (red perimeters). Receptors can be quantified by calculation of an expression index (EI = % +ve stained cells × mean fluorescence intensity in those cells) and proportional cell surface expression (PCSE) is calculated as the cell surface EI as a % of the whole cell EI. Panel C shows PCSE values calculated from the same representative experiment as used for panels A and B. Panel D shows the cell surface EI for HA-hGnRHR in control and IN3 pre-treated cells, stimulated for the indicated period with 10⁻⁷ M GnRH. Note that the agonist-induced reduction in cell surface hGnRHR expression is only evident in IN3 pre-treated cells.

Fig. 2

Peptide and non-peptide antagonist effects on GnRHR localisation. Panels A–C: HeLa cells transduced with Ad HA-hGnRHR, h.XGnRHR or A261K-hGnRHR were incubated ∼20 h in medium with the indicated concentration of IN3 with 0 (ctrl.) or 10⁻⁷ M cetrorelix (cet.) before determining the cell surface expression index, as above. Note that cetrorelix had no effect on HA-hGnRHR alone, but synergised with IN3 to increase cell surface expression of hGnRHR. Similar effects were seen in cells expressing the signalling-deficient A261K hGnRHR mutant, demonstrating that the IN3 and cetrorelix effects on cell surface hGnRHR expression are not dependent upon G-protein activation. Panel D: cells transduced with Ad HA-h.XGnRHR or hGnRHR were incubated for 60 min with anti-HA at 21 °C. They were then washed and incubated for 60 min with 10⁻⁷ M GnRH or cetrorelix, or without test compound (ctrl.) before fixation and staining (DAPI and anti-HA). In HA-h.XGnRHR expressing cells GnRH caused an increase in bright punctate anti-HA staining (indicating agonist-induced receptor internalisation into endosomes) and this was measured using a granularity assay to quantify “inclusions” (panel E). Note that inclusions were not measurable in HA-hGnRHR expressing cells because there are too few hGnRHR at the cell surface for efficient labelling during the low temperature loading period. However, GnRH caused a dose-dependent increase in inclusion count in HA-h.XGnRHR expressing cells, whereas cetrorelix had the opposite effect. The implication is that the peptide increases cell surface hGnRHR expression by slowing its internalisation from the cell surface.

Fig. 3

Spatiotemporal characteristics of GnRH and PDBu-stimulated ERK regulation revealed using an ERK knock-down and add-back model. Cells were transfected in 96-well plates with ERK1/2 siRNAs and transduced with Ad ERK2-GFP and Ad mGnRHR prior to stimulation with 10⁻⁶ M GnRH or PDBu for the times indicated. They were then fixed and stained before image acquisition and analysis for the calculation of whole-cell ppERK2 intensity (upper left panel) and the N:C ERK2-GFP ratio (lower left panel). Representative regions of cell images are also shown for DAPI, ERK2-GFP and ppERK2 in cells stimulated with 10⁻⁶ M GnRH or PDBu as indicated (right panels). Note that in spite of comparable initial responses appreciable levels of ppERK2 and nuclear retention of ERK2-GFP are only seen at 120 min in the PDBu stimulated cells (scale bars: 30 μm).

Fig. 4

Live cell imaging with varied GnRH pulse frequency. Panel A: HeLa cells were transduced with Ad-mGnRHR and Ad-NFAT2-EFP and treated with 0 or 10⁻⁷ M GnRH for 20 min, washed with ice-cold PBS, fixed with 4% PFA, permeabilised and stained with DAPI. The panel shows representative images of cells acquired in the DAPI (blue) and EFP (green) image channels, with an example of the automated image segmentation used to define perimeters of nuclei and cells. Scale bar: 30 μm. Panel B: cells transduced with Ad-mGnRHR, Ad-NLS-BFP and Ad-NFAT2-EFP were treated with 10⁻⁹ M GnRH for 5 min at 30 min intervals, hourly intervals, or every 2 h, as indicated. All the wells were subject to half hourly washes (grey rectangles) 5 min after GnRH or control addition. Digital images were acquired from live cells and used to calculate the nuclear:cytoplasmic (N:C) ratio which was normalised to the control value obtained at time 0 in each well. Note that integrative tracking (i.e. the saw-tooth response seen when responses have not returned to control values before repeat stimulation) occurred at the highest pulse frequency. Panel C shows the response to 30 min pulses of 10⁻⁹ M GnRH along with the response seen in cells receiving constant stimulation with 10⁻⁹ M GnRH throughout the 4 h experiment (dotted line) and the underlying [Ca²⁺]_i estimated using an established mathematical model for GnRH signalling (Washington et al., 2004).