Characterisation of the adiponectin receptors: The non-conserved N-terminal region of AdipoR2 prevents its expression at the cell-surface

Keshvari, Sahar; Rose, Felicity J.; Charlton, Hayley K.; Scheiber, Nicole L.; Webster, Julie A.; Kim, Yu Hee; Ng, Choaping; Parton, Robert G.; Whitehead, Jonathan P.

doi:10.1016/j.bbrc.2013.01.092

Cited by 14 publications

(31 citation statements)

References 30 publications

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“…We also demonstrated that this difference is due to the non-conserved N-terminal trunks of AdipoR1 and AdipoR2 200 . In the current report we have extended these studies by performing further characterisation of the molecular features governing the cell-surface expression and subsequent coupling to downstream signalling effectors of AdipoR1 and AdipoR2.…”

Section: Introductionmentioning

confidence: 74%

“…and AdipoR2 differed with around 50% of AdipoR1 present on the cell-surface whilst AdipoR2 was localised predominantly at the ER 200 . We subsequently demonstrated that the non-conserved, N-terminal trunks of AdipoR1 and AdipoR2 (1-81) underpinned these differences 200 .…”

Section: Serum Starvation Increases the Cell-surface Expression Of Admentioning

confidence: 93%

“…Quantitative measurement of total and cell-surface expression of AdipoR1 and AdipoR2 was performed, in permeabilised and non-permeabilised cells respectively, using a plate-based assay as described 200 . Briefly, parental HEK293 cells or transfected HEK293 cells were incubated in either the presence or absence of serum at 37°C overnight.…”

Section: Plate-based Determination Of Cell-surface Expression Of Adipmentioning

confidence: 99%

“…Immunofluorescence microscopy of permeabilised and non-permeabilised cells was performed as described 200 affording details of the subcellular distribution of the receptors and the cell-surface expression respectively. Images were taken using a Delta Vision OMX microscope (Applied Precision, GE Healthcare, Washington, USA).…”

Section: Immunofluorescence Microscopymentioning

confidence: 99%

“…AdipoR2 is more abundant in the ER 200 . We also demonstrated that this difference is due to the non-conserved N-terminal trunks of AdipoR1 and AdipoR2 200 .…”

Section: Introductionmentioning

confidence: 97%

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Molecular characterisation of the adiponectin receptors, AdipoR1 and AdipoR2

Keshvari¹

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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...

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Section: Introductionmentioning

confidence: 74%

Section: Serum Starvation Increases the Cell-surface Expression Of Admentioning

confidence: 93%

Section: Plate-based Determination Of Cell-surface Expression Of Adipmentioning

confidence: 99%

Section: Immunofluorescence Microscopymentioning

confidence: 99%

“…AdipoR2 is more abundant in the ER 200 . We also demonstrated that this difference is due to the non-conserved N-terminal trunks of AdipoR1 and AdipoR2 200 .…”

Section: Introductionmentioning

confidence: 97%

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