GnRH acts via G-protein coupled receptors to stimulate synthesis and secretion of luteinizing hormone and follicle-stimulating hormone. GnRH activates phospholipase C, causing calcium mobilisation and influx, activation of protein kinase C and stimulation of mitogen-activated protein kinase cascades. GnRH is secreted in brief pulses with each pulse causing rapid and transient activation of extracellular signal regulated kinases (ERKs) (1), yet very little is known about what shapes ERK responses to acute GnRH receptor activation. The cellular ERK concentration varies markedly between different cell types and individual cells (even of clonal cell lines). Here we have explored the possible relationship between ERK concentration and acute responses to GnRH. We have used a knock-down add-back protocol to firstly remove endogenous ERKs with siRNAs, and then reintroduce ERK2-GFP by means of an adenovirus. This allows us to monitor both total ERK levels (via the GFP) and activated ERK (using an antibody to phosphorylated ERK (ppERK)) with automated microscopy. We binned the individually imaged cells according to ERK2-GFP expression (mathematical sorting) and found that GnRH caused a rapid and transient increase in ppERK (maximal at 46 minutes) at approximately physiological ERK2-GFP levels. However, at higher ERK concentrations, the GnRH response was slower in onset (maximal at 1214 minutes). As a consequence of this difference, maximal ERK activation actually occurred at submaximal ERK concentrations but only at the early time points (2 and 4 minutes). A similar difference in response kinetics was observed when cells with high or low ERK2-GFP expression were physically sorted, by flow cytometry, before imaging. We conclude that ERK concentration can have a pronounced effect on activation kinetics (as well as amplitude) of ERK responses, that may be particularly relevant for acute responses such as those elicited by pulsatile hormonal stimulation. 1. Armstrong SP et al. (2010) J Biol Chem 285:2436071.
Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding: No specific grant from any funding agency in the public, commercial or not-for-profit sector.