Dynamin Is Required for GnRH Signaling to L-Type Calcium Channels and Activation of ERK

Abstract

We have shown that GnRH-mediated engagement of the cytoskeleton induces cell movement and is necessary for ERK activation. It also has previously been established that a dominant negative form of the mechano-GTPase dynamin (K44A) attenuates GnRH activation of ERK. At present, it is not clear at what level these cellular events might be linked. To explore this, we used live cell imaging in the gonadotrope-derived αT3-1 cell line to determine that dynamin-green fluorescent protein accumulated in GnRH-induced lamellipodia and plasma membrane protrusions. Coincident with translocation of dynamin-green fluorescent protein to the plasma membrane, we demonstrated that dynamin colocalizes with the actin cytoskeleton and the actin binding protein, cortactin at the leading edge of the plasma membrane. We next wanted to assess the physiological significance of these findings by inhibiting dynamin GTPase activity using dynasore. We find that dynasore suppresses activation of ERK, but not c-Jun N-terminal kinase, after exposure to GnRH agonist. Furthermore, exposure of αT3-1 cells to dynasore inhibited GnRH-induced cyto-architectural rearrangements. Recently it has been discovered that GnRH induced Ca(2+) influx via the L-type Ca(2+) channels requires an intact cytoskeleton to mediate ERK phosphorylation. Interestingly, not only does dynasore attenuate GnRH-mediated actin reorganization, it also suppresses Ca(2+) influx through L-type Ca(2+) channels visualized in living cells using total internal reflection fluorescence microscopy. Collectively, our data suggest that GnRH-induced membrane remodeling events are mediated in part by the association of dynamin and cortactin engaging the actin cytoskeleton, which then regulates Ca(2+) influx via L-type channels to facilitate ERK phosphorylation.

Gonadotropin releasing hormone activation of the mTORC2/Rictor complex regulates actin remodeling and ERK activity in LβT2 cells

“…Cells were fixed in 4% PFA and then stained with Alexa 488-conjugated phalloidin and DAPI and imaged by CLSM. Our results confirm our previous studies that actin remodeling occurs following GnRHa stimulation (Edwards et al, 2016; Navratil et al, 2014; Navratil et al, 2007). Consistent with mTORC1 not effecting ERK activity, pretreatment with RAPA did not affect actin reorganization.…”

“…Thus, understanding the mechanisms that GnRH utilizes to engage actin has important implications for fertility regulation. Previously, our group found that dynamin and cortactin are two key cell signaling intermediates that associate to engage the actin cytoskeleton following GnRH activation (Edwards, Dang, Murtazina et al, 2016; Navratil et al, 2014). Beyond this work, the full cohort of signaling mechanisms that GnRH utilizes to engage actin remodeling remains unclear.…”

Functional Role of Gonadotrope Plasticity and Network Organization

“…Consistent with this data, αT3-1 cells transfected with K44A resulted in a loss of GnRH-induced actin remodeling events ( 62 ). Our group also demonstrated that pharmacological inhibition of dynamin GTPase activity, using both dynasore and dyngo, not only perturbed GnRH-induced actin reorganization but also significantly suppressed ERK activation ( 63 ). Thus, highlighting the importance of dynamin GTPase activity in actin reorganization and subsequent MAPK activation.…”

Novel Insights into Gonadotropin-Releasing Hormone Action in the Pituitary Gonadotrope

“…28,29 Recent studies by Edwards and colleagues extended our understanding of the mechanisms of the role of actin cytoskeleton in GnRH signaling via L-type Ca 2þ channels and ERK activation. 30 Dynamin is a membrane-associated GTPase that is involved in membrane constriction, vesiculation, and cell wall formation. 31 Disrupting dynamin signaling in gonadotropes has long been known to disrupt GnRH signaling to the ERK pathway [32][33][34] ; however, the mechanistic basis for this has not been fully appreciated.…”

“…Actin cytoskeletal remodeling, necessary for gonadotropin secretion, is dependent on cortactin localization and these studies implicate dynamin signaling and calcium influx through Ltype Ca 2þ channels in this process. 30…”