Isolation and characterization of a bovine genomic clone encoding α_2D-adrenergic receptor - Possible role of amino acid residues in the third cytoplasmic loop in determining ligand binding

PURPOSE: To isolate a genomic clone for the bovine a2D adrenergic receptor (02D-AR) and analyze Hs structural and pharmacological properties. METHODS: A genomic clone for the bovine am-AR was isolated by screening a X EMBL3 library and its pharmacological properties were analyzed through ligand binding studies using transiently expressed wild type or mutant receptors. RESULTS: About 3kb including the coding region and putative TATA box was sequenced and the coding region was transiently expressed in COS7 cells followed by pharmacological analyses: The receptor bound rauwolscine with a Ko of 23 nM. ICso values for oxymetazoline, rauwolscine and prasozin were 100, 60 and 4000 nM respectively. These values, and the kinetics of binding, are in close agreement to the values determined for the a2crAR in photoreceptor cells. Its expression in retina was established through RNase protection assay. Mutation of five amino acid residues in the third cytoplasmic loop of the rat ot2oAR altered the ability of the receptor to bind to prazosin, epinephrine, and paminoclonidine but not to rauwolscine and phentolamine. CONCLUSIONS: A bovine genomic clone that is expressed in the retina has been isolated and encodes an cra-AR. Amino acid residues in the third cytoplasmic loop may play a role in determining the pharmacological properties of the receotor.