Received for publication September 5, 2006.
Revised October 23, 2006.
Accepted for publication October 24, 2006.

Comparison of signaling pathways activated by the relaxin family peptide receptors, RXFP1 and RXFP2, revealed using reporter genes

Abstract

The receptors for H2 relaxin and insulin-like peptide 3 (INSL3), RXFP1 and RXFP2 respectively, were recently identified and their signaling pathways are not yet well characterized. Although previous work has suggested that cAMP is a major signaling pathway activated by these receptors, RXFP1 has also been shown to activate a number of other signaling proteins. To this end, we examined the effect of stimulation of RXFP1 and RXFP2 receptors (expressed in HEK293T cells) with human relaxin family peptides upon a number of transcription factor response elements coupled to reporter genes. Hence reporter gene activity measured by enzyme activity in the cell media is a measure of the activation of a particular signaling pathway. A total of eight reporter genes were tested at both receptors, as a screen to identify other signaling pathways activated by RXFP1 and RXFP2. The cAMP response element reporter was strongly activated by both receptors. This effect was enhanced by pre-incubation with pertussis toxin (PTX), suggesting that Gs and inhibitory Gi/Go proteins mediate this response. Only activation of RXFP1 inhibited NFB transcription and this was reversed by PTX and the phosphoinositide-3- kinase inhibitor wortmannin. In addition, the glucorticoid response element was activated by RXFP1, but not by RXFP2 and was not activated in the parent HEK293T cells. Thus the use of reporter genes enabled differences in signaling between these two receptors to be revealed, that also throw light on the wide range of effects attributed to relaxin.

Key words: LGR7, LGR8, RXFP1, RXFP2, relaxin, reporter gene

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