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In collaboration with Adrian Chester (Vascular Biology), Dr Ginette Hoare is cloning cDNAs that represent novel splice variants of the human endothelin A receptor (ETAR) RNA. Bioinformatics analysis reveals the existence of several mRNA variants of ETAR that differ in their inclusion or exclusion of particular protein coding exons. Some of these are predicted to form truncated proteins that are unlikely to insert into the membrane or bind endothelin-1. Nevertheless, there is evidence that such truncated proteins may directly affect function of the wild-type receptor and downstream signalling. ETAR belongs to the extensive family of membrane-spanning G-protein coupled receptors (GPCRs) that mediate signal transduction to downstream kinases such as PI-3K. Another GPCR, Gonadotrophin-releasing hormone Receptor (GnRH-R) exists as full-length GnRH-R and also a C-terminal truncated protein created by alternative splicing of the GnRH-R transcript (Grosse et al , 1997). This shorter variant protein interferes with normal GnRH-R function and signal transduction. Co-expression of recombinant wild-type and truncated proteins in transiently or stably transfected COS-7 cells showed that the shorter variant localized to the membrane but did not bind agonist and was incapable of G protein-coupled signal transduction as measured by accumulation of labelled IP 3 . Crucially, co-expression revealed that this effect was specific for full-length GnRH-R, but not when the variant was co-expressed with other cloned GPCRs. Together, these data suggested that the variant GnRH-R can modify normal wild-type GnRH-R function. A possible physiological significance for variant isoform expression is demonstrated by the evidence of Kottler and colleagues (1999), who described a second GnRH-R splice variant and showed that the three mRNAs exhibit varied tissue-specific expression patterns in normal and malignant human tissues.
Our study, which is funded by the British Heart Foundation, will co-express cloned wild-type and variant ETAR isoforms in a cell line negative for ETAR expression to examine the effect of the variant isoforms upon wild-type G-protein coupled signal transduction and calcium signalling (in collaboration with Cell Electrophysiology).
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Further reading
Grosse R, Schoneberg T, Schultz G & Gudermann T (1997). Inhibition of gonadotropin-releasing hormone receptor signalling by expression of a splice variant of the human receptor. Mol. Endocr . 11, 1305-1318.
Kottler ML, Bergametti F, Carre MC, Morice S, Decoret E, Lagarde JP, Starzec A & Counis R (1999) Tissue-specific pattern of variant transcripts of the human gonadotropin-releasing hormone receptor gene. Eur J Endocrinol . 140, 561-9.
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