title: Differential trafficking of adenosine receptors in hippocampal neurons monitored using GFP- and super-ecliptic pHluorin-tagged receptors.
abstract: Adenosine receptors (ARs) modulate many cellular and systems-level processes in the mammalian CNS. However, little is known about the trafficking of ARs in neurons, despite their importance in controlling seizure activity and in neuroprotection in cerebral ischaemia. To address this we examined the agonist-dependent internalisation of C-terminal GFP-tagged A(1)Rs, A(2A)Rs and A(3)Rs in primary hippocampal neurons. Furthermore, we developed a novel super-ecliptic pHluorin (SEP)-tagged A(1)R which, via the N-terminal SEP tag, reports the cell-surface expression and trafficking of A(1)Rs in real-time. We demonstrate the differential trafficking of ARs in neurons: A(3)Rs internalise more rapidly than A1Rs, with little evidence of appreciable A(2A)R trafficking over the time-course of the experiments. Furthermore, the novel SEP-A(1)R construct revealed the time-course of internalisation and recovery of cell-surface expression to occur within minutes of agonist exposure and removal, respectively. These observations highlight the labile nature of A(1)R and A(3)Rs when expressed at the neuronal plasma membrane. Given the high levels of adenosine in the brain during ischaemia and seizures, internalisation of the inhibitory A(1)R may result in hyperexcitability, increased brain damage and the development of chronic epileptic states.
authors.affiliation: School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: A E
authors.initials: AE
authors.lastname: Baines
authors.forename: S A L
authors.initials: SA
authors.lastname: Corrêa
authors.forename: A J
authors.initials: AJ
authors.lastname: Irving
authors.forename: B G
authors.initials: BG
authors.lastname: Frenguelli
mesh_terms: D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism*; D006624:Hippocampus / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D009474:Neurons / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D058906:Purinergic P1 Receptor Agonists / Q000378:metabolism; D051381:Rats; D043682:Receptor, Adenosine A1 / Q000378:metabolism*; D043705:Receptor, Adenosine A2A / Q000378:metabolism*; D043684:Receptor, Adenosine A3 / Q000378:metabolism*; D018047:Receptors, Purinergic P1 / Q000378:metabolism
chemical_list: C485187:PHluorin; D058906:Purinergic P1 Receptor Agonists; D043682:Receptor, Adenosine A1; D043705:Receptor, Adenosine A2A; D043684:Receptor, Adenosine A3; D018047:Receptors, Purinergic P1; D049452:Green Fluorescent Proteins