MOLECULAR-CLONING, EXPRESSION AND CHARACTERIZATION OF CDNA-ENCODING AMOUSE ALPHA(1A)-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 se quence and amplified cDNA fragments from total RNA of mouse cerebral c ortex, liver and kidney by reverse transcription-polymerase chain reac tion (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 f rame of 1398 nucleotides encoding a 466 amino acid peptide which had 9 7%, 92% and 90% identity with the deduced amino acid sequences of the rat, human and bovine alpha(1a)-adrenoceptor, respectively. 3 The ampl ified mouse cDNA was inserted into a mammalian expression vector pcDNA 3.1(+) 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 fo r [H-3]-prazosin (K-d = 0.48 nM) and pattern of affinity for antagonis ts in competition studies that is similar to that of the rat alpha(1a) -adrenoceptor. Chloroethylclonidine (CEC) could slowly alkylate the ex pressed protein, with a rate similar to that of the rat alpha(1a)-adre noceptor. 4 The expressed receptors were able to mediate noradrenaline (NA) stimulation of the production of inositol phosphates in COS-I ce lls, consistent with coupling to phospholipase C. This response to NA could be reversed by pretreatment of the transfected cells with prazos in. 5 Based on the above evidence, we concluded that the cloned cDNA i s that of the mouse alpha(1a)-adrenoceptor.