Gonadotropin-releasing hormone receptors (GnRHRs) are expressed in gonadotropes and several extra-pituitary sites. They are assumed to be cell surface proteins but the human (h) GnRHR lacks features favoring plasma membrane localization and receptor location varies with cell type. When expressed in mammary (MCF7) cells, cell surface hGnRHR binding was much lower than that of mouse and sheep GnRHRs (type I GnRHRs without C-terminal tails), Xenopus (X) and marmoset type II GnRHRs (type II GnRHRs with C-tails) or chimeric receptors (type I GnRHRs with added XGnRHR C-tails). hGnRHR binding was higher in αT4 (gonadotrope-derived) cells and was increased less by C-tail addition. Whole cell levels of tagged human, Xenopus and chimeric GnRHRs were comparable (Western blotting) and confocal microscopy revealed that the hGnRHR is primarily intracellular (distribution similar to the endoplasmic reticulum marker, calreticulin), whereas most XGnRHR is at the plasma membrane, and adding the C-tail increased cell surface hGnRHR levels. A membrane-permeant antagonist increased cell surface hGnRHR number (>4-fold, t½ = 4 h) and also increased hGnRHR signaling and hGnRHR-mediated inhibition of proliferation. A more rapid increase in hGnRHR binding occurred when the temperature was raised from 4 to 37 °C (>5-fold, t½ = 15 min) and this effect was prevented by mutation to prevent signaling. Thus, cell surface GnRHR expression depends on receptor and cell type and the hGnRHR is primarily an intracellular protein that traffics to the cell surface for signaling in MCF7 cells. Manipulations favoring such trafficking may facilitate selective targeting of extra-pituitary GnRHRs.
Kathleen R Sedgley, Ann R Finch, Christopher J Caunt and Craig A McArdle
Ann R Finch, Kathleen R Sedgley, Christopher J Caunt and Craig A McArdle
In heterologous expression systems, human GnRH receptors (hGnRHRs) are poorly expressed at the cell surface and this may reflect inefficient exit from the endoplasmic reticulum. Here, we have defined the proportion of GnRHRs at the cell surface using a novel assay based on adenoviral transduction with epitope-tagged GnRHRs followed by staining and semi-automated imaging. We find that in MCF7 (breast cancer) cells, the proportional cell surface expression (PCSE) of hGnRHRs is remarkably low (<1%), when compared with Xenopus laevis (X) GnRHRs (∼40%). This distinction is retained at comparable whole cell expression levels, and the hGnRHR PCSE is increased by addition of the XGnRHR C-tail (h.XGnRHR) or by a membrane-permeant pharmacological chaperone (IN3). The IN3 effect is concentration- and time-dependent and IN3 also enhances the hGnRHR-mediated (but not h.XGnRHR- or mouse GnRHR-mediated) stimulation of [3H]inositol phosphate accumulation and the hGnRHR-mediated reduction in cell number. We also find that the PCSE for hGnRHRs and h.XGnRHRs is low and is greatly increased by IN3 in two hormone-dependent cancer lines, but is higher and less sensitive to IN3 in a gonadotrope line. Finally, we show that the effect of IN3 on hGnRHR PCSE is not mimicked or blocked by two peptide antagonists although they do increase the PCSE for h.XGnRHRs, revealing that an antagonist-occupied cell surface GnRHR conformation can differ from that of the unoccupied receptor. The low PCSE of hGnRHRs and this novel peptide antagonist effect may be important for understanding GnRHR function in extrapituitary sites.
Iain R Thompson, Annisa N Chand, Kim C Jonas, Jacky M Burrin, Mark E Steinhelper, Caroline P Wheeler-Jones, Craig A McArdle and Robert C Fowkes
In the pituitary, C-type natriuretic peptide (CNP) has been implicated as a gonadotroph-specific factor, yet expression of the CNP gene (Nppc) and CNP activity in gonadotrophs is poorly defined. Here, we examine the molecular expression and putative function of a local gonadotroph natriuretic peptide system. Nppc, along with all three natriuretic peptide receptors (Npr1, Npr2 and Npr3), was expressed in both αT3-1 and LβT2 cells and primary mouse pituitary tissue, yet the genes for atrial-(ANP) and B-type natriuretic peptides (Nppa and Nppb) were much less abundant. Putative processing enzymes of CNP were also expressed in αT3-1 cells and primary mouse pituitaries. Transcriptional analyses revealed that the proximal 50 bp of the murine Nppc promoter were sufficient for GNRH responsiveness, in an apparent protein kinase C and calcium-dependent manner. Electrophoretic mobility shift assays showed Sp1/Sp3 proteins form major complexes within this region of the Nppc promoter. CNP protein was detectable in rat anterior pituitaries, and electron microscopy detected CNP immunoreactivity in secretory granules of gonadotroph cells. Pharmacological analyses of natriuretic peptide receptor activity clearly showed ANP and CNP are potent activators of cGMP production. However, functional studies failed to reveal a role for CNP in regulating cell proliferation or LH secretion. Surprisingly, CNP potently stimulated the human glycoprotein hormone α-subunit promoter in LβT2 cells but not in αT3-1 cells. Collectively, these findings support a role for CNP as the major natriuretic peptide of the anterior pituitary, and for gonadotroph cells as the major source of CNP expression and site of action.