Molecular cloning, expression and characterization of cDNA encoding a mouse alpha1a-adrenoceptor.
1. In this study, we have cloned, expressed, and characterized an alpha1a-adrenoceptor gene from the mouse. We designed oligonucleotide PCR primers complementary to regions of the rat alpha1a-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 alpha1a-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 alpha1a-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 [3H]-prazosin (Kd = 0.48 nM) and pattern of affinity for antagonists in competition studies that is similar to that of the rat alpha1a-adrenoceptor. Chloroethylclonidine (CEC) could slowly alkylate the expressed protein, with a rate similar to that of the rat alpha1a-adrenoceptor. 4. 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. 5. Based on the above evidence, we concluded that the cloned cDNA is that of the mouse alpha1a-adrenoceptor.
Xiao
L
L
Department of Pharmacology, Creighton University School of Medicine, Omaha, NE 68178, USA.
Scofield
M A
MA
Jeffries
W B
WB
D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D003000:Clonidine / Q000031:analogs & derivatives / Q000494:pharmacology; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D005260:Female; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011869:Radioligand Assay; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
C547713:ADRA1A protein, human; C547714:Adra1a protein, mouse; C547715:Adra1a protein, rat; D000317:Adrenergic alpha-Antagonists; D017931:DNA Primers; D018076:DNA, Complementary; D007295:Inositol Phosphates; D018340:Receptors, Adrenergic, alpha-1; C051731:chlorethylclonidine; D003000:Clonidine