Molecular cloning, expression and characterization of cDNA encoding a mouse α1a-adrenoceptor

1 In this study, we have cloned, expressed, and characterized an alpha(1a)-adrenoceptor gene from the mouse. We designed oligonucleotide PCR primers complementary to regions of the rat alpha(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). 2 Both the nucleotide and deduced peptide sequences of the cDNA showed high sequence identity with those of cloned alpha(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 alpha(1a)-adrenoceptor, respectively. 3 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 [H-3]-prazosin (K-d = 0.48 nM) and pattern of affinity for antagonists in competition studies that is similar to that of the rat alpha(1a)-adrenoceptor. Chloroethylclonidine (CEC) could slowly alkylate the expressed protein, with a rate similar to that of the rat alpha(1a)-adrenoceptor. 4 The expressed receptors were able to mediate noradrenaline (NA) stimulation of the production of inositol phosphates in COS-I cells, consistent with coupling to phospholipase C. This response to NA could be reversed by pretreatment of the transfected cells with prazosin. 5 Based on the above evidence, we concluded that the cloned cDNA is that of the mouse alpha(1a)-adrenoceptor.