Cloning and expression of a human endothelin receptor: subtype A.
The polymerase chain reaction, employing degenerate primers specific for the intramembrane domains III and VI of G-coupled receptors, was used to generate partial clones encoding those receptors carried by cultured rat aorta smooth muscle cells. One clone, spanning the intramembrane domains IV-VI of a receptor specific for endothelin-1 (ET-R[A]), was used as a probe to screen a human placental cDNA library. The clone pL4-3, encoding a selective type of human endothelin receptor (ET-R[A]), has an open reading frame encoding a protein 427 amino acids in length, with a relative molecular weight of 48,625 daltons. The sequence analysis suggests the presence of a signal peptide, two potential sites for glycosylation in the N terminal extracellular domain, the seven transmembrane domains typical of G-protein receptors, and several potential sites for phosphorylation in the C terminal cytoplasmic domain. At the amino acid level, the human ET-R(A) shows 91% and 94% identity with the rat and bovine ET-R(A)s, respectively, and 59% similarity with the human ET-R(B). Xenopus laevis oocytes injected with the cloned cDNA express binding sites specific for endothelin-1. Expression of the message in COS 7 cells gave a membrane-bound product to which binding of the [125I]-ET-1 was inhibited by peptide analogues specific for ET-R(A).
Hayzer
D J
DJ
Division of Cardiology, Emory University School of Medicine, Atlanta, Georgia 30322.
Rose
P M
PM
Lynch
J S
JS
Webb
M L
ML
Kienzle
B K
BK
Liu
E C
EC
Bogosian
E A
EA
Brinson
E
E
Runge
M S
MS
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000502:physiology; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D016232:Endothelins / Q000378:metabolism*; D005260:Female; D015723:Gene Library; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000502:physiology; D009838:Oligodeoxyribonucleotides; D010920:Placenta / Q000502:physiology; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D051381:Rats; D017466:Receptors, Endothelin / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D016232:Endothelins; D009838:Oligodeoxyribonucleotides; D017466:Receptors, Endothelin