The Pitcher Lab

Pitcher lab member - Kathleen.jpg

KATHLEEN WEBB: Investigating a role for β-arrestin at the Golgi

b-Arrestins are best known for their role in mediating G
protein-coupled receptor (GPCR) desensitisation, at least in part, through
their ability to bind components of the endocytic machinery, including AP2,
clathrin and Arf6, thereby promoting receptor internalisation. I am
investigating a novel intracellular membrane trafficking role for β-arrestin 1 at
the Golgi. Mouse embryonic fibroblasts (MEFs) derived from β-arrestin 1
knockout (KO), but not β-arrestin 2 KO or wild type (WT) mice, exhibit a
fragmented Golgi morphology, a phenotype that is rescued upon β-arrestin 1
expression. Following the trafficking of ssDsRed in β-arrestin 1 KO as compared
to WT MEFs shows a faster rate of anterograde secretory trafficking in the
absence of β-arrestin 1. These observations suggest a role for
b-arrestin 1 in attenuating trafficking and the associated vesiculation
that occurs coincident with traffic flow through the Golgi. Co-immunoprecipitation
studies reveal that
b-arrestin 1 binds to the clathrin
adaptor AP1 and Arf1. Notably, in marked contrast to the previously
characterised role for Src in promoting dissociation of β-arrestin 1/AP2
complexes at the plasma membrane here we show here that Src potentiates
β-arrestin 1/AP1 complex formation. High Src activity is associated with rapid
Golgi transport rates and with a fragmented Golgi phenotype. It is thus
tempting to speculate that β-arrestin 1 may serve to attenuate anterograde
trafficking in a Src-dependent fashion. Notably, the fragmented Golgi
morphology observed in the HT29 colorectal cancer cell line, which has high
endogenous levels of active Src, is compacted by overexpression of
b-arrestin1, but not β-arrestin 2. We are currently assessing a
variety of biochemical secretory trafficking assays that will be used to probe
the role of
b-arrestin1 in regulating secretory traffic in more detail. It is
anticipated that the ability of
b-arrestin1
mutant constructs to regulate traffic flux may provide information concerning
the molecular players involved in this process. How Golgi morphology is maintained
in the face of continual and varied traffic flux is a fundamental problem in
cell biology, our results point to a role of
b-arrestin1 in this process.

 

Pitcher lab member - Katrina.jpg

KATRINA LESTER: Regulation of Class I HDAC function by GRK5

G-protein
coupled receptor kinase 5 (GRK5), known for its role in G-protein coupled receptor
(GPCR) desensitisation, can also adopt a nuclear localisation. Nuclear GRK5 has
been shown to have a causative role in pathological cardiac hypertrophy when
overexpressed in the hearts of transgenic (TG) mice. Hypertrophy is a
cardiovascular disease that represents a milestone in the progression to heart
failure. At the cellular level, pathological cardiac hypertrophy is concomitant
with the aberrant expression of foetal cardiac genes and the upregulation of
the prohypertrophic transcription factor, MEF2. This aberrant cardiac
remodelling ensues following pathological hypertrophic stimuli, which signal
through Gq coupled G-protein coupled receptors (GPCRs) to activate multiple
signalling pathways many of which converge upon histone deacetylase (HDAC)
regulation.

HDACs play a dual
role in cardiac hypertrophy; with class I (HDAC1, 2, 3 and 8) acting in a
prohypertrophic manner while class IIa (HDAC4, 5, 7 and 9) are
antihypertrophic. Class II HDACs are GRK5 substrates and their phosphorylation
relieves the inhibition of MEF2, thus resulting in the activation of
hypertrophy.

Here we demonstrate
a novel and direct interaction between GRK5 and the class I HDACs, with the
exception of HDAC2. Additionally, we show that GRK5 can also interact
indirectly with both HDAC1 and 2 via its direct binding to the transcriptional
repressor protein Sin3A. Considering that HDAC1 and GRK5 function to activate
hypertrophy, we hypothesize that both proteins may function in the same
prohypertrophic signalling pathway, potentially via the Sin3A repressor complex.
We have used GRK5 peptide arrays to map the binding sites of HDAC1 and Sin3A on
GRK5. It is anticipated that GRK5 mutant constructs lacking the ability to bind
HDAC1, Sin3A or both proteins will allow an assessment of the role of these
interactions in mediating pathological hypertrophy both in rat neonatal
ventricular myocytes in vitro and TG
mice in vivo. Our results suggest
that GRK5 may be potentiating cardiac hypertrophy via two distinct mechanisms;
GRK5-mediated phosphorylation and nuclear export of HDAC5 and also via a
GRK5/HDAC1/Sin3A transcriptional repressor complex. Patients with ventricular
overload disease have high cardiac levels of GRK5 expression. Elucidation of
the molecular pathways by which GRK5 is causing cardiac hypertrophy could
potentially lead to the development of novel specific inhibitors.

Pitcher lab member - James.jpg

JAMES ROBINSON: Identification of GRK2 as a Rho-activated MAPK scaffold

James has recently left the lab and is now a Post-doctoral Research Assistant with Dr. Patricia McDonald at The Scripps Research Institute, Florida.

The classical function of the GRKs is to desensitise GPCRs.
Subsequent work has demonstrated that the GRKs can also regulate
signalling from other receptors, including receptor tyrosine kinases
(RTKs). I have found that GRK2 interacts directly with the GTP-bound,
activated form of the small GTPase Rho. The GRK2 catalytic domain
mediates binding to Rho but GRK2 catalytic activity is not affected by
Rho binding. Rather, Rho binding to GRK2 specifically promotes GRK2
binding to Raf, MEK and ERK, the components of the ERK MAPK cascade.
Over-expression of GRK2 potentiates EGF-induced ERK activation in
HEK-293 cells by acting as a scaffold protein for the ERK MAPK cascade.
This novel scaffolding function of GRK2 is dependent on active Rho. I am
currently investigating the role of Rho-mediated ERK MAPK scaffolding
by GRK2 in the biology of vascular smooth muscle cells (VSMCs), where I
have found that GRK2 is required for EGF-mediated proliferation. We are
also interested in the effect of Rho binding on the function of the
other GRKs. I have found that all of the GRKs bind to Rho but ERK
scaffolding as a consequence of Rho binding is specific to GRK2. Perhaps
the other GRKs may act as scaffolds for other MAPK cascades in response
to Rho binding.