MOLECULAR-CLONING AND CHARACTERIZATION OF THE MAJOR ENDOTHELIN RECEPTOR SUBTYPE IN PORCINE CEREBELLUM

Endothelin receptors (ETRs) display subtype heterogeneity and are wide ly distributed throughout the tissues of the periphery and central ner vous system. In order to gain further insight into the potential molec ular differences of ETRs, we initiated molecular cloning of ETR genes by screening for the appearance of I-125-ET-1 binding activity in COS cells transfected with pools of a porcine cerebellum cDNA expression l ibrary. Two independent clones (pPCETR 1.1 and pPCETR 5.6) were identi fied and isolated by repeated rounds of pool enrichment and COS cell e xpression. 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 residue s in length and consists of seven potential transmembrane domains, wit h homology to members of the GTP-binding protein-coupled receptor supe rfamily. Northern analysis indicated a single mRNA species of about 5 kilobases, which is expressed significantly in cerebellum, lung, kidne y, and pituitary. Expression of functional receptor was demonstrated b y endothelin-1 (ET-1)-mediated Ca2+ mobilization in COS cells transfec ted with pPCETR 1.1 (COS/ETR 1.1) and ET-1-mediated electrophysiologic al responses in Xenopus oocytes injected with RNA derived from pPCETR 1.1. Quantitative comparison of saturation binding of I-125-ET-1 to ei ther porcine cerebellum or COS/ETR 1.1 membranes indicated an identica l apparent dissociation constant. The relative efficacy of ET-related peptides to compete for binding of I-125-ET-1 to receptor from porcine cerebellum and COS/ETR 1.1 indicated that both preparations encode a nonselective or ET(B)R subtype. Chemical cross-linking of I-125-ET-1 t o receptor derived from cerebellum or COS/ETR 1 revealed two bands, wi th apparent molecular masses of 47 and 35 kDa. These data demonstrate that the pPCETR 1.1 encodes the major ETR subtype in the porcine cereb ellum.