TY - JOUR
T1 - Palmitoylation of the adiponectin receptors, AdipoR1 and AdipoR2, is essential for function in vitro and in vivo
AU - Keshvari, Sahar
AU - Adams, Mark
AU - Henstridge, Darren
AU - O'Neill, Hayley M.
AU - Hooper, John
AU - Febbraio, Mark A.
AU - Whitehead, Jonathan P
PY - 2019/1
Y1 - 2019/1
N2 - Dysregulation of the adiponectin axis contributes
to obesity-related cardiometabolic disorders making
it an attractive therapeutic target. However,
our understanding of the adiponectin receptors,
AdipoR1 and AdipoR2, atypical seven transmembrane
domain proteins, is rudimentary. We reasoned
that elaboration of key properties of AdipoR1
and AdipoR2 would reveal therapeutic strategies.
To address this we have employed in silico, molecular
and cellular approaches.
First, using a series of complementary qualitative
(microscopy) and quantitative (flow cytometry)
assays we demonstrated that under steady-state
conditions (no serum starvation) AdipoR1 exhibits
robust (60%) cell-surface expression (CSE), whereas
AdipoR2 is predominantly restricted to the ER
[1]. Second, overexpression of AdipoR1 in HEK-293
cells resulted in acute activation of downstream
signalling networks (AMPK, AKT, ERK and P38MAPK)
whereas overexpression of AdipoR2 promoted more
chronic activation (peaking at 15 min and 24 h)
[2]. Third, characterisation of chimeric receptors
(comprised of a series of AdipoR1/R2 and
AdipoR2/R1 constructs) demonstrated that the differences
in CSE and temporal signalling profiles
of AdipoR1 and AdipoR2 are underpinned by the
non-conserved regions (spanning AdipoR1(1—70) and
AdipoR2(1—81)) in the cytoplasmic ‘trunks’ of the
receptors. Fourth, bioinformatics analysis (using
CSS-Palm) revealed several putative palmitoylation
sites including a conserved ‘canonical’ site (common
to GPCRs) in the juxtamembrane region of both
receptors as well as additional non-conserved sites.
Palmitoylation of these sites was confirmed using
Acyl-Biotinyl exchange chemistry and site-directed
mutagenesis which also revealed rapid turnover of
palmitoylation (t1/2 < 60 min). Moreover, palmitoylation
of the canonical site in AdipoR1(Cys124) or AdipoR2(Cys135) was required for efficient CSE and
coupling to downstream signalling networks (all
p < 0.05).
Collectively these findings demonstrate fundamental
differences between AdipoR1 and AdipoR2,
highlight the importance of the cytoplasmic
‘trunks’ and post-translational regulation (palmitoylation)
of the receptors. Studies are ongoing to
elaborate whether changes in the latter contribute
to the pathophysiology of cardiometabolic disease
and afford novel therapeutic opportunities.
AB - Dysregulation of the adiponectin axis contributes
to obesity-related cardiometabolic disorders making
it an attractive therapeutic target. However,
our understanding of the adiponectin receptors,
AdipoR1 and AdipoR2, atypical seven transmembrane
domain proteins, is rudimentary. We reasoned
that elaboration of key properties of AdipoR1
and AdipoR2 would reveal therapeutic strategies.
To address this we have employed in silico, molecular
and cellular approaches.
First, using a series of complementary qualitative
(microscopy) and quantitative (flow cytometry)
assays we demonstrated that under steady-state
conditions (no serum starvation) AdipoR1 exhibits
robust (60%) cell-surface expression (CSE), whereas
AdipoR2 is predominantly restricted to the ER
[1]. Second, overexpression of AdipoR1 in HEK-293
cells resulted in acute activation of downstream
signalling networks (AMPK, AKT, ERK and P38MAPK)
whereas overexpression of AdipoR2 promoted more
chronic activation (peaking at 15 min and 24 h)
[2]. Third, characterisation of chimeric receptors
(comprised of a series of AdipoR1/R2 and
AdipoR2/R1 constructs) demonstrated that the differences
in CSE and temporal signalling profiles
of AdipoR1 and AdipoR2 are underpinned by the
non-conserved regions (spanning AdipoR1(1—70) and
AdipoR2(1—81)) in the cytoplasmic ‘trunks’ of the
receptors. Fourth, bioinformatics analysis (using
CSS-Palm) revealed several putative palmitoylation
sites including a conserved ‘canonical’ site (common
to GPCRs) in the juxtamembrane region of both
receptors as well as additional non-conserved sites.
Palmitoylation of these sites was confirmed using
Acyl-Biotinyl exchange chemistry and site-directed
mutagenesis which also revealed rapid turnover of
palmitoylation (t1/2 < 60 min). Moreover, palmitoylation
of the canonical site in AdipoR1(Cys124) or AdipoR2(Cys135) was required for efficient CSE and
coupling to downstream signalling networks (all
p < 0.05).
Collectively these findings demonstrate fundamental
differences between AdipoR1 and AdipoR2,
highlight the importance of the cytoplasmic
‘trunks’ and post-translational regulation (palmitoylation)
of the receptors. Studies are ongoing to
elaborate whether changes in the latter contribute
to the pathophysiology of cardiometabolic disease
and afford novel therapeutic opportunities.
U2 - 10.1016/j.orcp.2016.10.032
DO - 10.1016/j.orcp.2016.10.032
M3 - Meeting Abstract
SN - 1871-403X
VL - 13
SP - 30
JO - Obesity Research and Clinical Practice
JF - Obesity Research and Clinical Practice
IS - 1
ER -
