Abstract
Hirschsprung's disease (HSCR) is one the most common congenital intestinal disease. It leads to aganglionic megacolon in the early childhood. Several susceptibility genes have been identified : RET protooncogene and its ligand, glial cell derived neutrophic factor (GDNF), Sox 10, Endothelin-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 been explored and some of them seem to be non fonctional variants. The properties of three mutant human endothelin B receptor (hETB) (G57S, R319W and P383L) in isolated HSCR were analyzed. Stable recombinant cells expressing the three mutants and the wild-type (WT) were established. The hETB receptors were characterized for 125I ET-1 binding, ET-1 induced signaling: calcium transient, AP-1 transcriptional factor activation and cAMP accumulation. Immunofluorescence experiments showed normal cellular distributions of the mutant G57S, R319W and WT hETB receptors. In contrast, the P383L hETB mutant receptor was concentrated near the nucleus and essentially no ET-1 binding was detected. The two other mutants (G57S and R319W) bound ET-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 hETB mutant even stimulated cAMP accumulation which was blocked by pertussis toxin. The absence of the P383L mutant receptor from the membrane clearly indicates that this mutation could be involved in HSCR. The G57S and R319W mutant receptors, despite their normal coupling to Gaq, have a defect in the Galphai signaling pathway and the G57S mutation couples to Galphas. These observations allow us to hypothesize that cAMP signaling might be involved in the differenciation of neural cells in the bowel.
Highlights
Hirschsprung’s disease (HSCR) is a frequent congenital intestinal malformation affecting 1 in 5000 live births [1], characterized by the absence of ganglion cells in the distal portion of the intestinal tract
We investigated the relationship between the G57S, R319W and P383L human endothelin B receptor (hETB) mutant receptors described by Amiel et al [19] and HSCR
Affinities of the Wild-Type and Mutant hETB Receptors for ET-1 We looked for the implication of these mutations in a predisposition to HSCR by examining their affinities for ET-1
Summary
Affinities of the Wild-Type and Mutant hETB Receptors for ET-1 We looked for the implication of these mutations in a predisposition to HSCR by examining their affinities for ET-1. The wild-type hETB receptor in CHO-æquorin cells was activated by endothelin to produce a dosedependent intracellular calcium transient (Fig. 4). Cells transfected with the P383L hETB receptor produced no intracellular calcium transient when stimulated with ET-1, in agreement with its poor binding of ET-1 and its location (Fig. 2). ET-1 (Fig. 5) and the specific ETB agonist, IRL 1620 (insert panel Fig. 5) bound to the wild-type or the mutant G57S or R319W hETB receptors and caused the same luciferase activity, indicating the same stimulation of the AP-1 pathway. ET-1 inhibited adenylate cyclase in a dose dependent manner on CHO cells transiently transfected to produce the wild-type hETB receptor This occurred after stimulation with 102 5 M forskolin (data not shown) and in basal conditions (Fig. 6). Pertussis toxin has no effect on the ET-1 blockade of adenylyl cyclase production via the wild-type hETB receptor (Fig. 7B)
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