Truncated human endothelin receptor A produced by alternative splicing and its expression in melanoma.

Yf Zhang,S Jeffery,Jc Kaski,Pa Berry,Sa Burchill,Nd Carter

doi:10.1038/bjc.1998.643

Abstract

In this study, reverse transcriptase polymerase chain reaction was used to amplify human endothelin receptor A (ETA) and ETB receptor mRNA. A truncated ETA receptor transcript with exons 3 and 4 skipped was found. The skipping of these two exons results in 109 amino acids being deleted from the receptor. The truncated receptor was expressed in all tissues and cells examined, but the level of expression varied. In melanoma cell lines and melanoma tissues, the truncated receptor gene was the major species, whereas the wild-type ETA was predominant in other tissues. A 1.9-kb ETA transcript was identified in melanoma cell lines by Northern blot, which was much smaller than the transcript in heart and in other tissues reported previously (4.3 kb). The cDNA coding regions of the truncated and wild-type ETA receptors were stably transfected into Chinese hamster ovary (CHO) cells. The truncated ETA receptor-transfected CHO cells did not show binding affinity to endothelin 1 (ET-1) or endothelin 3 (ET-3). The function and biological significance of this truncated ETA receptor is not clear, but it may have regulatory roles for cell responses to ETs.

Highlights

Chinese hamster ovary (CHO) cells were obtained from the American Culture Collection. [I']ET-l and ['1-I]endothelin 3 (ET-3) were purchased from Amersham International
A truncated endothelin receptor A (ETA) mRNA transcript in which exon 3 and exon 4 have been skipped was identified by Reverse transcription polymerase chain reaction (RT-PCR) and is most probably produced by alternative pre-mRNA splicing
In the constitutive pre-mRNA splicing of ETA receptor genes. all introns are spliced out and eig ht exons are subsequently connected in order. producing wild-type ETA mRNA

Summary

Methods

Superscript II reverse transcriptase xxas obtained from Gibco-BRL Life Technology. RNasin RNase inhibitor Awas purchased from the Promega Biotech Corporation. AmpliTaq and Cycle Sequencing kits were purchased from Perkin Elmer Cetus. The Lipofectin reagent for transfection was obtained from GibcoBRL Life Technology. Chinese hamster ovary (CHO) cells were obtained from the American Culture Collection. [I']ET-l and ['1-I]ET-3 were purchased from Amersham International. RNA preparation Total RNA was extracted from frozen tissues or cultured cell lines with the single-step RNA extraction method The quality of RNA was evaluated by checkincg the integrity of 28S and 18S ribosome bands after electrophoresis. The concentration of RNA samples was measured by combining optical densitv at 260 nm and staining, intensity and comparinga these with.

Results

Discussion

Conclusion