The application of the human beta-globin gene locus control region and murine erythroleukemia cell system to the expression and pharmacological characterization of human endothelin receptor subtypes.
The cDNAs encoding both A and B subtypes of the human endothelin receptor have been inserted into mammalian cell expression vectors that utilize the human globin gene, locus control region. These constructs have been introduced into murine erythroleukemia cells and inducible high level expression of the receptors has been achieved (approximately 1.5-pM/mg membrane protein and approximately 13,500 binding sites/cell for both receptor subtypes). Cell lines expressing these receptors were obtained on a rapid time scale (3-4 weeks), facilitated by the need for the analysis of only small numbers of cell clones/receptor (approximately 6). Competitive binding assays with endothelin-1 gave IC50s of 130 +/- 30 pM for endothelin-A receptor and 160 +/- 30 pM for endothelin-B receptor. Similar studies with the different isoforms of endothelin, sarafatoxin-S6b and -S6c, BQ123 and BQ3020, all gave the expected selectivity profiles. The IC50s for all compounds were in close agreement with those reported for native receptors. Thus, this expression system, which has several advantages over other described expression systems, is capable of rapidly providing large quantities of receptor for detailed pharmacological analyses or drug screening. In addition, the expressed receptors display the expected pharmacological profiles in the absence of any complicating, competing interactions from other subtypes or binding sites.
Davies
A
A
Cardiovascular and Metabolism Research Department, Zeneca Pharmaceuticals, Macclesfield, Cheshire, UK.
Whiting
E
E
Bath
C
C
Tang
E
E
Brennand
J
J
D001667:Binding, Competitive; D002452:Cell Count; D018076:DNA, Complementary; D016232:Endothelins / Q000494:pharmacology*; D015870:Gene Expression; D005914:Globins / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D011869:Radioligand Assay; D017466:Receptors, Endothelin / Q000096:biosynthesis* / Q000145:classification / Q000235:genetics*
D018076:DNA, Complementary; D016232:Endothelins; D017466:Receptors, Endothelin; D005914:Globins