Relaxin signalling links tyrosine phosphorylation to phosphodiesterase andadenylyl cyclase activity

The relaxin receptor has so far avoided molecular cloning and characterizat ion. We have therefore characterized the signalling events activated by rel axin (RLX), using two different cell culture-based bioassay systems: primar y human endometrial stromal cells from the cycle (ESC) and the human monocy te cell line THP-1. Upon RLX stimulation, both cell types showed a rapid in crease in cAMP accumulation, which could be inhibited by an inhibitor of G- protein activation, GDP-beta -S. However, evolutionarily one would expect t he RLX receptor, like those for the structurally related hormones insulin a nd insulin-like growth factor-I, to involve tyrosine kinase activity. The s pecific tyrphostins AG 1478, AG 527 and AG 879 inhibited the RLX-stimulated cAMP response in human ESC and THP-1 cells in a dose-dependent manner, tho ugh the potent broad range tyrphostin AG 213 had no effect. Also, treatment of THP-1 cells with the potent phosphotyrosine phosphatase inhibitors bpV( phen) and mpV(pic) increased RLX-stimulated cAMP accumulation in a dose-dep endent manner. The effect of the general tyrosine kinase inhibitor genistei n (which can also inhibit some phosphodiesterases) on RLX-mediated cAMP acc umulation strongly depended on the activity status of phosphodiesterase. In the absence of a phosphodiesterase inhibitor, genistein enhanced RLX-stimu lated cAMP accumulation in both bioassays. When phosphodiesterase was inhib ited by isobutylmethylxanthine, this effect was not observed. The results i mply that activation of the RLX receptor uses tyrosine kinase signalling to control phosphodiesterase activity, and hence to up-regulate intracellular cAMP.