Oral Presentation Australian & New Zealand Obesity Society 2015 Annual Scientific Meeting

New insights into the Adiponectin Receptors: AdipoR1 and AdipoR2 show different cell surface expression and temporal signalling profiles and both are dependent on palmitoylation. (#41)

Sahar Keshvari 1 , Mark Adams 2 , John Hooper 1 , Jon Whitehead 1
  1. Mater Research_UQ, Woolloongabba, QLD, Australia
  2. QUT, Brisbane, QLD, Australia

Dysregulation of the adiponectin axis contributes to obesity-related cardiometabolic disorders making it an obvious 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 ER1. Second, overexpression of AdipoR1 in HEK-293 cells resulted in acute activation of downstream signalling networks (AMPK, AKT, ERK & 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.
  1. Keshvari, S. et al. Biochem Biophys Res Commun 432, 28-33, (2013).
  2. Keshvari, S. and Whitehead, J. P. Mol Cell Endocrinol 409, 121-9, (2015).