Molecular cloning and characterization of the major endothelin receptor subtype in porcine cerebellum.
Endothelin receptors (ETRs) display subtype heterogeneity and are widely distributed throughout the tissues of the periphery and central nervous system. In order to gain further insight into the potential molecular differences of ETRs, we initiated molecular cloning of ETR genes by screening for the appearance of 125I-ET-1 binding activity in COS cells transfected with pools of a porcine cerebellum cDNA expression library. Two independent clones (pPCETR 1.1 and pPCETR 5.6) were identified and isolated by repeated rounds of pool enrichment and COS cell expression. DNA sequence analysis of pPCET 1.1 and pPCET 5.6 indicated that both clones have the same nucleotide sequence; the deduced amino acid sequence indicated that the porcine cerebellum ETR is 443 residues in length and consists of seven potential transmembrane domains, with homology to members of the GTP-binding protein-coupled receptor superfamily. Northern analysis indicated a single mRNA species of about 5 kilobases, which is expressed significantly in cerebellum, lung, kidney, and pituitary. Expression of functional receptor was demonstrated by endothelin-1 (ET-1)-mediated Ca2+ mobilization in COS cells transfected with pPCETR 1.1 (COS/ETR 1.1) and ET-1-mediated electrophysiological responses in Xenopus oocytes injected with RNA derived from pPCETR 1.1. Quantitative comparison of saturation binding of 125I-ET-1 to either porcine cerebellum or COS/ETR 1.1 membranes indicated an identical apparent dissociation constant. The relative efficacy of ET-related peptides to compete for binding of 125I-ET-1 to receptor from porcine cerebellum and COS/ETR 1.1 indicated that both preparations encode a nonselective or ETBR subtype. Chemical cross-linking of 125I-ET-1 to receptor derived from cerebellum or COS/ETR 1 revealed two bands, with apparent molecular masses of 47 and 35 kDa. These data demonstrate that the pPCETR 1.1 encodes the major ETR subtype in the porcine cerebellum.
Elshourbagy
N A
NA
Department of Molecular Genetics, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
Lee
J A
JA
Korman
D R
DR
Nuthalaganti
P
P
Sylvester
D R
DR
Dilella
A G
AG
Sutiphong
J A
JA
Kumar
C S
CS
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D001667:Binding, Competitive; D015152:Blotting, Northern; D015139:Blotting, Southern; D002118:Calcium / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism*; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004247:DNA / Q000235:genetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D011869:Radioligand Assay; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D017466:Receptors, Endothelin; D013552:Swine; D014018:Tissue Distribution; D014158:Transcription, Genetic; D014982:Xenopus laevis
D003432:Cross-Linking Reagents; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D017466:Receptors, Endothelin; D004247:DNA; D002118:Calcium