A non-olfactory shark adenosine receptor activates CFTR with unique pharmacology and structural features

Adenosine receptors (ADORs) are G-protein coupled purinoceptors that have several functions including regulation of chloride secretion via CFTR in human airway and kidney. We cloned an ADOR from Squalus acanthias (shark) that likely regulates CFTR in the rectal gland. Phylogenic- and expression- analyses indicate that elasmobranch ADORs are non-olfactory, and appear to represent extant predecessors of mammalian ADORs. We therefore designate the shark ADOR as the A 0 receptor. We co-expressed A 0 with CFTR in Xenopus laevis oocytes and characterized the coupling of A 0 to the chloride channel. Two electrode voltage clamping was performed and current-voltage (I-V) responses were recorded to monitor CFTR status. Only in A 0 - and CFTR- co-injected oocytes did adenosine analogs produce a significant concentration-dependent activation of CFTR consistent with its electrophysiological signature. A pharmacological profile for A 0 was obtained for ADOR agonists and antagonists that differed markedly from all mammalian ADOR subtypes (agonists: R-PIA > S-PIA > CGS21680 > CPA > 2ClADO > CV1808 = DPMA > NECA) and (antagonists: DPCPX > PD115199 > 8PT > CGC > CGS15943). Structures of human ADORs permitted a high-confidence homology model of the shark A 0 core which revealed unique structural features of ancestral receptors. We conclude: (1) A 0 is a novel and unique adenosine receptor ancestor by functional and structural criteria; (2) A 0 likely activates CFTR in vivo and this receptor activates CFTR in oocytes indicating an evolutionary coupling between ADORs and chloride secretion; and (3) A 0 appears to be a non-olfactory evolutionary ancestor of all four mammalian ADOR subtypes. Significance Statement We have cloned and characterized an ancient adenosine receptor from sharks that is unlikely to be olfactory in function. The shark receptor, which we designate as A 0 , has a unique pharmacological profile, characteristic structural features, and is also highly likely to be the dominant ADOR regulator of the shark ancient ortholog of the Cystic Fibrosis chloride channel, called CFTR.