The adiponectin axis is a major regulator of metabolic, cardiovascular and inflammatory tone. Current understanding of the adiponectin receptors, R1 and R2, is rudimentary constraining our ability to target these atypical seven trans-membrane receptors. Hence, we have started to characterise R1 and R2. We previously reported that R1 exhibits robust (60%) cell-surface expression (CSE) under steady-state conditions (no serum starvation) whilst R2 is undetectable and showed these differences were predicated by the non-conserved N-terminal, intracellular trunks1.
Here we hypothesised that (i) CSE of R1/R2 is regulated, (ii) R1/R2 are subject to palmitoylation and this regulates CSE/function (as in GPCRs). To address these hypotheses we have employed bioinformatics, cell-based and in vivo approaches.
We demonstrate that (i) serum starvation increases R2 CSE and adiponectin stimulates internalisation of R1/R2 (all p<0.05). (ii) Bioinformatics analysis revealed a putative conserved ‘canonical’ palmitoylation site in the juxtamembrane region of R1 (124) and R2 (135), plus additional non-conserved sites. This was confirmed using mutagenesis/acyl-biotinyl exchange chemistry and palmitoylation of R1(124) or R2(135) was shown to be required for efficient CSE of R1 and R2 and (iii) for coupling to ERK and P38MAPK in cell-based systems. We were unable to detect adiponectin-stimulated AMPK phosphorylation in vitro so performed in vivo electrotransfer to examine the effects of WT or palmitoylation deficient (R1(C124A)/R2(C135A)) constructs on AMPK phosphorylation in mouse skeletal muscle. Overexpression of WT R1 but not R1(C124A) increased AMPK phosphorylation (2-fold, p<0.05). Neither WT R2 or R2(C135A) promoted AMPK phosphorylation.
These results provide increasing evidence that CSE of R1 and R2 is under complex, differential regulation and is essential for coupling adiponectin to downstream signalling effectors. Further studies are required to elaborate the detailed molecular mechanisms that may provide novel therapeutic opportunities.