Hetero-oligomerization between adenosine A1 and P2Y1 receptors in living cells: Formation of ATP-sensitive adenosine receptors

✍ Scribed by Hiroyasu Nakata; Kazuaki Yoshioka; Osamu Saitoh

• Publisher: John Wiley and Sons
• Year: 2003
• Tongue: English
• Weight: 272 KB
• Volume: 58
• Category: Article
• ISSN: 0272-4391
• DOI: 10.1002/ddr.10169

✦ Synopsis

Abstract

We explored the possibility of hetero‐oligomerization between G~i/o~ protein‐coupled A~1~ adenosine receptor (A~1~R) and G~q~ protein‐coupled P2Y~1~ receptor (P2Y~1~R) in transfected cultured cells by a new bioluminescence resonance energy transfer technology (BRET^2^) in addition to indirect biochemical or pharmacological methods. The existence of A~1~R/P2Y~1~R hetero‐oligomers in cotransfected HEK293T cells was first shown using coimmunoprecipitation methods. In the same cotransfected cells, ADPβS was able to reduce forskolin‐evoked cAMP accumulation in a pertussis toxin‐ and A~1~R antagonist‐sensitive manner, indicating that the pharmacology of A~1~R was significantly modified in the cotransfected cells, i.e., ADPβS binds A~1~R and inhibits adenylyl cyclase activity via G~i/o~ proteins. Also, a high degree of A~1~R and P2Y~1~R colocalization was demonstrated in cotransfected cells by double immunofluorescence experiments with confocal laser microscopy. Then the BRET^2^ technique revealed constitutive heteromeric oligomerization between A~1~R and P2Y~1~R in living HEK293T cells. The BRET^2^ signal also increased in a time‐dependent manner upon addition of agonists for both receptors, which was inhibited by pretreatment with the P2Y~1~R antagonist MRS2179, indicating that this process is promoted by the simultaneous activation of both receptors. These results suggest that the oligomeric association of A~1~R with P2Y~1~R generates A~1~R with P2Y~1~R‐like agonistic pharmacology and provides a molecular mechanism for an increased diversity of purinergic signaling. Existence of this hybrid purinergic receptor may explain the controversial inhibition of synaptic transmission by adenine nucleotides. Drug Dev. Res. 58:340–349, 2003. © 2003 Wiley‐Liss, Inc.