A nonolfactory 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 cystic fibrosis transmembrane conductance regulator (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 nonolfactory and appear to represent extant predecessors of mammalian ADORs. We therefore designate the shark ADOR as the A₀ receptor. We coexpressed A₀ with CFTR in Xenopus laevis oocytes and characterized the coupling of A₀ to the chloride channel. Two-electrode voltage clamping was performed, and current-voltage (I-V) responses were recorded to monitor CFTR status. Only in A₀- and CFTR-coinjected oocytes did adenosine analogs produce a significant concentration-dependent activation of CFTR consistent with its electrophysiological signature. A pharmacological profile for A₀ was obtained for ADOR agonists and antagonists that differed markedly from all mammalian ADOR subtypes [agonists: R-phenyl-isopropyl adenosine (R-PIA) > S-phenyl-isopropyl adenosine (S-PIA) > CGS21680 > N⁶-cyclopentyladenosine (CPA) > 2-chloroadenosine (2ClAdo) > CV1808 = N⁶-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine (DPMA) > N-ethyl-carboxyl adenosine (NECA); and antagonists: 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > PD115199 > 1,3-dimethyl-8-phenylxanthine (8PT) > CGS15943]. Structures of human ADORs permitted a high-confidence homology model of the shark A₀ core that revealed unique structural features of ancestral receptors. We conclude that 1) A₀ is a novel and unique adenosine receptor ancestor by functional and structural criteria; 2) A₀ likely activates CFTR in vivo, and this receptor activates CFTR in oocytes, indicating an evolutionary coupling between ADORs and chloride secretion; and 3) A₀ appears to be a nonolfactory evolutionary ancestor of all four mammalian ADOR subtypes.