Genomic organization and expression of the human alpha 1B-adrenergic receptor.
alpha 1-Adrenergic receptors (ARs) are members of the guanine nucleotide-binding protein-coupled receptor superfamily. The genes for all ARs described thus far are intronless. We report here the cloning and the nucleotide sequence of the gene for the human alpha 1B-AR. It consists of two exons and a single large intron of at least 20 kilobases which interrupts the coding region at the end of the putative sixth transmembrane domain. The deduced amino acid sequence of the encoded receptor has a high degree of homology to the cloned hamster, rat, and dog alpha 1B-ARs. To characterize the encoded protein, a fusion gene constructed by splicing together exon 1 and exon 2 was expressed transiently in COS-1 cells. The transfected gene fusion product resulted in the production of an alpha 1B-AR with ligand binding characteristics indistinguishable from those of the expressed hamster alpha 1B cDNA. Evidence that the human alpha 1B-AR gene we have isolated is indeed transcribed is the finding of similar sized (2.8-kilobase) transcripts in human heart and other tissues by Northern blot analysis when either exon 1 or exon 2 is used as a probe. Moreover, using primers designed to span the exon 1/exon 2 boundary, a polymerase chain reaction product generated from single-stranded DNA prepared from human heart mRNA had the exact size and nucleotide sequence predicted for a transcript in which exon 1 is spliced to exon 2. The 5'-flanking region (924 base pairs (bp)) of exon 1 contains neither a TATA box nor a CAAT box but is high in GC content (70%) and contains several Sp1 binding sites (GC boxes), consistent with promoters described for housekeeping genes. The 5'-untranslated region also contains a putative cyclic AMP response element. Primer extension studies and RNase protection assays suggested that there are several potential transcription start sites in most tissues with a predominant site located 173 bp upstream from the translation start site. The 3'-flanking region contains a putative polyadenylation signal (ATTAAA) 492 bp downstream from the stop codon. The genomic organization of the human alpha 1B-AR with a single large intron interrupting its coding region differs from those of other ARs as well as muscarinic and 5-hydroxy-tryptamine receptors, which are intronless. The location of the intron in the human alpha 1B-AR gene is also unique among those members of the G-protein-coupled receptor family that do possess introns.(ABSTRACT TRUNCATED AT 400 WORDS)
Ramarao
C S
CS
Department of Heart and Hypertension Research, Cleveland Clinic Foundation, Ohio 44195.
Denker
J M
JM
Perez
D M
DM
Gaivin
R J
RJ
Riek
R P
RP
Graham
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004247:DNA; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D011942:Receptors, Adrenergic, alpha / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D012333:RNA, Messenger; D011942:Receptors, Adrenergic, alpha; D004247:DNA