P2Y1and P2Y12 receptor heterodimerisation: from recombinant systems to native detection

Mohammed Adnan A Safar, Rachel Aimee Wood (Editor), Rothwelle Tate (Editor), Margaret Rose Cunningham, Charles Kennedy (Editor)

Research output: Contribution to conferenceSpeech

Abstract

Purinergic P2Y1 and P2Y12 receptors are G protein-coupled receptors (GPCRs) that have been demonstrated to dimerise with other GPCRs. Our unpublished studies suggest that hP2Y1 and hP2Y12 heterodimerise therefore the aim of this study was to investigate the functional relevance of receptor interaction, firstly in recombinant systems and then natively in microglial cells.

tSA201 cells, which are a transformed human kidney cell line, were transfected or co-transfected with hP2Y1 and hP2Y12 receptors. Their cellular co-localisation was determined using confocal microscopy and physical interaction by co-immunoprecipitation. Cell surface expression and receptor trafficking was quantified using surface ELISA approach. Native P2Y receptors were detected in mouse BV-2 microglial cells using RT-PCR and indirect immunofluorescence.

Previous co-immunoprecipitation experiments revealed that P2Y1 and P2Y12 receptors heterodimerisation. This study shows that hP2Y1 and hP2Y12 receptors express predominantly at the cell surface in tSA201 cells. hP2Y12 receptor expression was reduced by 22.5% (n=3), at the cell surface when co-expressed with hP2Y1, but not vice-versa. hP2Y1 and hP2Y12 internalization to ADP (0-60mins, 10µM) was maximum 30 min post-treatment (28.8% ±6.2 and 20.8% ±7.8, respectively). When receptors were co-expressed, ADP did not reduce receptor surface expression (n=3). Interestingly, native P2Y1 and P2Y12 receptors in BV-2 cells also appear to co-localise with Pearson's correlation coefficient =0.684 ±0.004 (n=3, 200 cells), however signals were detected intracellularly rather than at the cell surface.

hP2Y1 and hP2Y12 receptor heterodimersation impacted ADP-mediated internalisation when both receptors were overexpressed in tSA201 cells. Co-localisation studies in BV-2 cells suggest that the location of receptor interaction may differ depending upon the cell types explored. Further work is under way to investigate these differences.
Original languageEnglish
Publication statusPublished - 4 Dec 2018
EventBPS-MPGPCR 2018 - Monash University, Parkville Campus, Melbourne, Australia
Duration: 2 Dec 20184 Dec 2018
https://www.monash.edu/pharm/about/events/BPS-MPGPCR-Meeting-2018

Conference

ConferenceBPS-MPGPCR 2018
CountryAustralia
CityMelbourne
Period2/12/184/12/18
Internet address

Keywords

  • heterodimerisation
  • receptor heterodimerisation
  • Purinergic P2Y1 receptors
  • Purinergic P2Y12 receptors

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