The role of filamin in gonadotropin releasing hormone receptor signalling and expression
Date
2020
Authors
Motlogelwa, Lawrence Katlego
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Gonadotropin Releasing Hormone (GnRH) is the central regulator of reproductive
development and function. When GnRH binds to its receptor it results in signal
transduction cascades which eventually culminate in gonadotropin synthesis and
secretion. These hormones in turn regulate steroidogenesis and gametogenesis.
The GnRH receptor can activate various signalling pathways in pituitary
gonadotropes, however the Gαq/11 protein kinase C (PKC) dependent extracellular
signal-regulated (ERK) pathway remains the main pathway through which
gonadotropin synthesis and secretion are mediated. The mechanisms underlying
the GnRH receptor ERK pathway have not been fully defined, it is not clear how
signal specificity of this pathway is maintained. One of the explanations is the
presence of scaffolding proteins and signalling complexes (signalosomes), which
have been shown to involve the cytoskeleton and associated proteins.
Filamin A, an actin-binding cytoskeletal protein has been shown to modulate
trafficking and signalling of several receptors. Filamin A organises complex
cellular signals from the plasma membrane, cytoplasm to the nucleus. Filamin A
has also been shown to interact with upstream effectors of ERK, namely PKC and
mitogen activated protein kinase kinase (MEK 1). However, the role of filamin A
in GnRH receptor signalling has not yet been studied. We investigated the role of
filamin A in GnRH receptor signalling. Filamin A siRNAs were designed and
synthesised to reduce filamin A protein expression in GnRH receptor-transfected
HEK 293 cells, then GnRH receptor-mediated ERK phosphorylation was
measured by western blotting. There was no significant difference in HEK 293
cells with normal and reduced protein expression of filamin A. GnRH receptor mediated ERK phosphorylation was also measured in GnRH receptor-transfected
filamin A replete (M2A7) and deficient (M2) melanoma cells. GnRH receptor mediated ERK phosphorylation kinetics varied between M2A7 and M2 cells.
ERK phosphorylation in the M2 cells reached its maximum after 15 minutes and
started to decrease after 30 minutes. In contrast ERK phosphorylation in the
M2A7 cells was elevated from 1 minute and sustained through to 60 minutes.
Gonadotropin Releasing Hormone (GnRH) is the central regulator of reproductive
development and function. When GnRH binds to its receptor it results in signal
transduction cascades which eventually culminate in gonadotropin synthesis and
secretion. These hormones in turn regulate steroidogenesis and gametogenesis.
The GnRH receptor can activate various signalling pathways in pituitary
gonadotropes, however the Gαq/11 protein kinase C (PKC) dependent extracellular
signal-regulated (ERK) pathway remains the main pathway through which
gonadotropin synthesis and secretion are mediated. The mechanisms underlying
the GnRH receptor ERK pathway have not been fully defined, it is not clear how
signal specificity of this pathway is maintained. One of the explanations is the
presence of scaffolding proteins and signalling complexes (signalosomes), which
have been shown to involve the cytoskeleton and associated proteins.
Filamin A, an actin-binding cytoskeletal protein has been shown to modulate
trafficking and signalling of several receptors. Filamin A organises complex
cellular signals from the plasma membrane, cytoplasm to the nucleus. Filamin A
has also been shown to interact with upstream effectors of ERK, namely PKC and
mitogen activated protein kinase kinase (MEK 1). However, the role of filamin A
in GnRH receptor signalling has not yet been studied. We investigated the role of
filamin A in GnRH receptor signalling. Filamin A siRNAs were designed and
synthesised to reduce filamin A protein expression in GnRH receptor-transfected
HEK 293 cells, then GnRH receptor-mediated ERK phosphorylation was
measured by western blotting. There was no significant difference in HEK 293
cells with normal and reduced protein expression of filamin A. GnRH receptor mediated ERK phosphorylation was also measured in GnRH receptor-transfected
filamin A replete (M2A7) and deficient (M2) melanoma cells. GnRH receptor mediated ERK phosphorylation kinetics varied between M2A7 and M2 cells.
ERK phosphorylation in the M2 cells reached its maximum after 15 minutes and
started to decrease after 30 minutes. In contrast ERK phosphorylation in the
M2A7 cells was elevated from 1 minute and sustained through to 60 minutes.
Gonadotropin Releasing Hormone (GnRH) is the central regulator of reproductive
development and function. When GnRH binds to its receptor it results in signal
transduction cascades which eventually culminate in gonadotropin synthesis and
secretion. These hormones in turn regulate steroidogenesis and gametogenesis.
The GnRH receptor can activate various signalling pathways in pituitary
gonadotropes, however the Gαq/11 protein kinase C (PKC) dependent extracellular
signal-regulated (ERK) pathway remains the main pathway through which
gonadotropin synthesis and secretion are mediated. The mechanisms underlying
the GnRH receptor ERK pathway have not been fully defined, it is not clear how
signal specificity of this pathway is maintained. One of the explanations is the
presence of scaffolding proteins and signalling complexes (signalosomes), which
have been shown to involve the cytoskeleton and associated proteins.
Filamin A, an actin-binding cytoskeletal protein has been shown to modulate
trafficking and signalling of several receptors. Filamin A organises complex
cellular signals from the plasma membrane, cytoplasm to the nucleus. Filamin A
has also been shown to interact with upstream effectors of ERK, namely PKC and
mitogen activated protein kinase kinase (MEK 1). However, the role of filamin A
in GnRH receptor signalling has not yet been studied. We investigated the role of
filamin A in GnRH receptor signalling. Filamin A siRNAs were designed and
synthesised to reduce filamin A protein expression in GnRH receptor-transfected
HEK 293 cells, then GnRH receptor-mediated ERK phosphorylation was
measured by western blotting. There was no significant difference in HEK 293
cells with normal and reduced protein expression of filamin A. GnRH receptor mediated ERK phosphorylation was also measured in GnRH receptor-transfected
filamin A replete (M2A7) and deficient (M2) melanoma cells. GnRH receptor mediated ERK phosphorylation kinetics varied between M2A7 and M2 cells.
ERK phosphorylation in the M2 cells reached its maximum after 15 minutes and
started to decrease after 30 minutes. In contrast ERK phosphorylation in the
M2A7 cells was elevated from 1 minute and sustained through to 60 minutes.
Furthermore, ligand affinity and cell surface receptor expression were assessed
using competition binding assays, effector coupling was assessed by measuring
GnRH receptor-mediated IP production using IP assays. Filamin A is not required
for GnRH receptor-mediated ERK phosphorylation, but its presence results in
rapid and sustained GnRH receptor-mediated ERK phosphorylation. Furthermore,
the latter is not due to changes in GnRH ligand binding affinity, cell surface
receptor expression or G protein coupling. These results suggest that filamin A is
part of the GnRH receptor signalosome and is required for enhanced ERK
phosphorylation, thus filamin A may have a role in the regulation of reproductive
development and function.
Description
A dissertation submitted in fulfilment of the requirements for the degree of Master of Science in Medicine to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, 2020