Cloning and expression of a rat brain alpha 2B-adrenergic receptor.
We have isolated a cDNA clone (RB alpha 2B) and its homologous gene (GR alpha 2B) encoding an alpha 2B-adrenergic receptor subtype by screening a rat brain cDNA and a rat genomic library. Nucleotide sequence analysis showed that both clones code for a protein of 458 amino acids, which is 87% homologous to the human kidney glycosylated adrenergic receptor (alpha 2-C4) and divergent from the rat kidney nonglycosylated alpha 2B subtype (RNG alpha 2). Transient expression of RB alpha 2B in COS-7 cells resulted in high-affinity saturable binding (Kd = 0.25 nM) for [3H]rauwolscine and a high receptor number (Bmax = 7.7 pmol/mg of protein) in the membranes of transfected COS-7 cells. Pharmacological analysis demonstrated that the expressed receptor bound adrenergic ligands with the following order of potency: rauwolscine greater than yohimbine greater than prazosin greater than oxymetazoline, with a prazosin-to-oxymetazoline Ki ratio of 0.34. This profile is characteristic of the alpha 2B-adrenergic receptor subtype. Blotting analysis of rat brain mRNA gave one major (3.0-kilobase) and two minor (4.6- and 2.3-kilobase) mRNA species, and hybridization with strand-specific probes showed that both DNA strands of GR alpha 2B may be transcriptionally active. These findings show that rat brain expresses an alpha 2B-adrenergic receptor subtype that is structurally different from the rat kidney nonglycosylated alpha 2B subtype. Thus the rat expresses at least two divergent alpha 2B-adrenergic receptors.
Flordellis
C S
CS
Section of Hypertension and Atherosclerosis, Boston University School of Medicine, MA 02118.
Handy
D E
DE
Bresnahan
M R
MR
Zannis
V I
VI
Gavras
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D015698:Genomic Library; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011061:Poly A / Q000235:genetics; D012313:RNA / Q000235:genetics; D015347:RNA Probes; D012333:RNA, Messenger; D051381:Rats; D011942:Receptors, Adrenergic, alpha / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D015016:Yohimbine / Q000378:metabolism
D015347:RNA Probes; D012333:RNA, Messenger; D011942:Receptors, Adrenergic, alpha; D011061:Poly A; D015016:Yohimbine; D012313:RNA; D004247:DNA