Molecular cloning, expression and characterization of cDNA encoding a mouse α_1a-adrenoceptor

• In this study, we have cloned, expressed, and characterized an α_1a-adrenoceptor gene from the mouse. We designed oligonucleotide PCR primers complementary to regions of the rat α_1a-adrenoceptor sequence and amplified cDNA fragments from total RNA of mouse cerebral cortex, liver and kidney by reverse transcription-polymerase chain reaction (RT–PCR).

• Both the nucleotide and deduced peptide sequences of the cDNA showed high sequence identity with those of cloned α_1a-adrenoceptors from other species. The cDNA clone had an open reading frame of 1398 nucleotides encoding a 466 amino acid peptide which had 97%, 92% and 90% identity with the deduced amino acid sequences of the rat, human and bovine α_1a-adrenoceptor, respectively.

• The amplified mouse cDNA was inserted into a mammalian expression vector pcDNA3.1(+) and expressed in COS-1 cells. The pharmacological properties of the mouse cDNA clone were examined in radioligand binding studies and functional assays. The expressed mouse protein had a high affinity for [³H]-prazosin (K_d=0.48 nM) and pattern of affinity for antagonists in competition studies that is similar to that of the rat α_1a-adrenoceptor. Chloroethylclonidine (CEC) could slowly alkylate the expressed protein, with a rate similar to that of the rat α_1a-adrenoceptor.

• The expressed receptors were able to mediate noradrenaline (NA) stimulation of the production of inositol phosphates in COS-1 cells, consistent with coupling to phospholipase C. This response to NA could be reversed by pretreatment of the transfected cells with prazosin.

• Based on the above evidence, we concluded that the cloned cDNA is that of the mouse α_1a-adrenoceptor.