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Cloning and Functional Identification of Novel Endothelin Receptor Type A Isoforms in Pituitary

Noriyuki Hatae, Nadia Aksentijevich, Hana W. Zemkova, Karla Kretschmannova, Melanija Tomi and Stanko S. Stojilkovic

Section on Cellular Signaling, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, Maryland 20892-4510

Address all correspondence and requests for reprints to: Stanko S. Stojilkovic, Section on Cellular Signaling, National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Building 49, Room 6A-36, 49 Convent Drive, Bethesda, Maryland 20892-4510. E-mail: stankos{at}helix.nih.gov.

Mammalian endothelin (ET) receptors, termed ET_AR and ET_BR, are derived from two intron-containing genes and the functional splice variants of ET_BR but not ET_AR have been identified. Here, we report about the isolation of cDNAs of ET_AR transcripts from rat anterior pituitary, which are generated by alternative RNA splicing. Deletion of exon 2 and insertion of fragments from intron 1 and 2 accounted for formation of three misplaced proteins, whereas the insertion of a fragment from intron 6 resulted in generation of a functional plasma membrane receptor, termed ET_AR-C13. In this splice variant, the C-terminal 382S-426N sequence of ET_AR was substituted with a shorter 382A-399L sequence, resulting in alteration of the putative domains responsible for coupling to G_q/11 and G_s proteins and the endocytotic recycling, as well as in deletion of the predicted protein kinase C/casein kinase 2 phosphorylation sites. The mRNA transcripts for ET_AR-C13 were identified in normal and immortalized pituitary cells and several other tissues. The pharmacological profiles of recombinant ET_AR and ET_AR-C13 were highly comparable, but the coupling of ET_AR-C13 to the calcium-mobilizing signaling pathway was attenuated, causing a rightward shift in the potency for agonist. Furthermore, the efficacy of ET_AR-C13 to stimulate adenylyl cyclase signaling pathway and to internalize was significantly reduced. These results indicate for the first time the presence of a novel ET_A splice receptor, which could contribute to the functional heterogeneity among secretory pituitary cell types.