Molecular characterisation of the adiponectin receptors, AdipoR1 and AdipoR2

Abstract

The increasing global prevalence of cardiovascular and metabolic diseases necessitates the development of more effective therapeutic strategies that, in turn, require a greater understanding of the regulatory networks involved. Research over the last decade has increased our appreciation of the key role of the adiponectin axis as a major regulator of metabolic, cardiovascular and inflammatory tone, thereby establishing it as a province of therapeutic opportunity. The receptors for adiponectin, AdipoR1 and AdipoR2, are distant relatives of the largest single class of drug targets, the G-protein coupled receptor (GPCR) family. However, unlike GPCRs they have intracellular N-termini and extracellular C-termini and signal via atypical pathways. Our current understanding of AdipoR1 and AdipoR2 is rudimentary, constraining our ability to target these receptors effectively. The aim of this thesis was to characterise molecular features of AdipoR1 and AdipoR2 that facilitate adiponectin signal transduction to advance our understanding and identify strategies to enhance adiponectin's beneficial effects.We have begun to characterise basic properties of AdipoR1 and AdipoR2, focusing on molecular factors that drive cell-surface expression (CSE) of the receptors using a range of C-terminal, epitope-tagged AdipoR1 and AdipoR2 constructs. Surprisingly, under steady-state conditions (no serum starvation) only AdipoR1 was readily detected on the cell-surface (cell-surface ratio of AdipoR1 vs AdipoR2 is 0.6±0.1 vs 0.15±0.1, p<0.05). Generation and characterisation of a series of chimeric and truncated constructs demonstrated that a non-conserved, intracellular, N-terminal region of AdipoR2 (R2 ) restricted its CSE whilst the same region in AdipoR1 (R1 (1-70) ) promoted its CSE. We also confirmed that AdipoR1 and AdipoR2 form heterodimer and that coexpression of these receptors increase the CSE of AdipoR2. Subsequently, we provided evidence that the subcellular localisation of AdipoR1 and AdipoR2 is governed by multiple motifs across their non-conserved and conserved cytoplasmic domains. For instance, two highly conserved motifs, an ER exit motif (FxxxFxxxF) and Di-Leucine motif (DxxxLL), in the conserved Nterminal domain are required for the proper CSE of both AdipoR1 and AdipoR2, whilst different parts of the non-conserved domain of AdipoR2 inhibits its CSE.Moreover, we demonstrated that 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). This difference was also underpinned by the non-conserved N-terminal domains of AdipoR1 and AdipoR2. We also demonstrated that a number of conserved and non-conserved cysteines in the N-terminal domain of iii AdipoR1 and AdipoR2 are subject to palmitoylation and that palmitoylation of a conserved cysteine, situated in the juxta-membrane region of the N-termini of AdipoR1 and AdipoR2 in a position analogous to t...