The adiponectin axis regulates cardiometabolic tone making it an attractive therapeutic focus. However, strategies to target the receptors, AdipoR1 and AdipoR2, are limited by a rudimentary understanding of these atypical proteins. We reasoned that elaboration of key properties of AdipoR1 and AdipoR2 would reveal therapeutic strategies. To address this we employed a combination of 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, in HEK 293 cells over-expressing AdipoR1 adiponectin activated downstream signalling networks (AMPK, AKT, ERK & P38MAPK) acutely (peaking at 15 min) whereas signal transduction via AdipoR2 was relatively chronic (peaking at 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 in the trunks 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 (t_1/2 < 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.