Functional characterization of three mutations of the endothelin B receptor gene in patients with Hirschsprung's disease: Evidence for selective lossof G(i) coupling

Background: Hirschsprung's disease (HSCR) is one the most common congenital intestinal disease. It leads to aganglionic megacolon in the early childho od. Several susceptibility genes have been identified : RET protooncogene a nd its ligand, glial cell derived neutrophic factor (GDNF), Sox 10, Endothe lin-3 (EDN3) and its receptor B (EDNRB). EDNRB mutations are found in 5% of familial or sporadic HSCR. Only few EDNRB mutations found in HSCR have bee n explored and some of them seem to be non fonctional variants. Materials and Methods: The properties of three mutant human endothelin B re ceptor (hET(B)) (G57S, R319W and P383L) in isolated HSCR were analyzed. Sta ble recombinant cells expressing the three mutants and the wild-type (WT) w ere established. The hET(B) receptors were characterized for I-125 ET-1 bin ding, ET-1 induced signaling : calcium transient, AP-1 transcriptional fact or activation and cAMP accumulation. Results: Immunofluorescence experiments showed normal cellular distribution s of the mutant G57S, R319W and WT hET(B) receptors. In contrast, the P383L hET(B) mutant receptor was concentrated near the nucleus and essentially n o ET-1 binding was detected. The two other mutants (G57S and R319W) bound E T-1 normally, induced calcium transients and activated the AP-1 pathway in the same way as wild type, but did not inhibit adenylate cyclase. The G57S hET(B) mutant even stimulated cAMP accumulation which was blocked by pertus sis toxin. Conclusion The absence of the P383L mutant receptor from the membrane clear ly indicates that this mutation could be involved in HSCR. The G57S and R31 9W mutant receptors, despite their normal coupling to G(alphaq) have a defe ct in the G(alphai) signaling pathway and the G57S mutation couples to G(al phas). These observations allow us to hypothesize that cAMP signaling might be involved in the differenciation of neural cells in the bowel.