Novel Mutations of the Endothelin B Receptor Gene in Patients with Hirschsprung's Disease and Their Characterization

Abstract

Hirschsprung's disease (HSCR) is a congenital intestinal disease, characterized by the absence of ganglion cells in the distal portion of the intestinal tract. Recently, three susceptibility genes have been identified in HSCR, namely the RET protooncogene, the endothelin B (ET B ) receptor gene (EDNRB), and the endothelin-3 (ET-3) gene (EDN3). To investigate whether mutations in EDNRB could be related with HSCR in non-inbred populations in Japan, we examined alterations of the gene in 31 isolated patients. Three novel mutations were detected as follows: two transversions, A to T and C to A at nucleotides 311 (N104I) and 1170 (S390R), respectively, and a transition, T to C at nucleotide 325 (C109R). To analyze functions of these mutant receptors, they were expressed in Chinese hamster ovary cells. S390R mutation did not change the binding affinities but caused the decreases in the ligand-induced increment of intracellular calcium and in the inhibition of adenylyl cyclase activity, showing the impairment of the intracellular signaling. C109R receptors were proved to be localized near the nuclei as an unusual 44-kDa protein with the extremely low affinity to endothelin-1 (ET-1) and not to be translocated into the plasma membrane. On the other hand, N104I receptors showed almost the same binding affinities and functional properties as those of the wild type. Therefore, we conclude that S390R and C109R mutations could cause HSCR but that N104I mutation might be polymorphous. Hirschsprung's disease (HSCR)1 is a congenital intestinal disorder, characterized by the absence of ganglion cells in the distal portion of the intestinal tract, as a consequence of premature arrest of cranio-caudal migration of neural crest cells. Recently, three susceptibility genes have been identified in HSCR, namely the RET protooncogene, the endothelin B (ET B ) receptor gene (EDNRB), and the endothelin-3 (ET-3) gene (EDN3).Mice with targeted null disruption of Ret exhibited an autosomal recessive phenotype comprising the total lack of the enteric nervous system and renal agenesis (1), that provided an excellent model of HSCR. Approximately 50 mutations in RET have so far been identified, and the RET mutations account for 50% of familial and 15-20% of sporadic cases of HSCR, over 75% of which are associated with long segment HSCR (2-6).Endothelins (ETs) are a family of 21 amino acid vasoactive peptides, consisting of three isopeptides, ET-1, ET-2 and ET-3, that act on G protein-coupled receptors, ET A and ET B (7-11). ET A exhibits different affinities to the three isopeptides in the order of ET-1 Ն ET-2 Ͼ Ͼ ET-3. ET B accepts all three isopeptides equally. These receptors regulate multiple effector pathways, for example, phospholipase C via G q , and adenylyl cyclase via G s in smooth muscle cells (ET A ), and via G i in endothelial cells (ET B ) (12, 13). Mice with targeted null disruption of either Ednrb or Edn3 exhibit an identical phenotype, coat color spotting, and aganglionic megacolon, similar to HSCR or Shah-Waardenburg syndrome in...

Characterization of a family of endogenous neuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8

“…LtkϪ cells, stably cotransfected with the human GPR7 cDNA and the Gqi chimeric G protein ␣ subunit cDNA, were used for the cytoplasmic Ca 2ϩ transient assay (13). Cytoplasmic calcium levels were monitored by using Fura-2͞AM as described (14). In situ hybridization on mouse brain slices was performed as described (15).…”

G Protein-Coupled Receptor Trafficking in Health and Disease: Lessons Learned to Prepare for Therapeutic Mutant Rescue in Vivo

“…Information condensed as follows: rhodopsin (Stojanovic and Hwa, 2002;Mendes et al, 2005;Tao, 2006); V2R (Bernier et al, 2004a,b;Fujiwara and Bichet, 2005;Bichet, 2006;Boson et al, 2006;Robben and Deen, 2007;Robben et al, 2006); GnRHR (Beranova et al, 2001;Janovick et al, 2003a;Ulloa-Aguirre et al, 2004a,b); CaR (Brown, 2007;Huang and Breitwieser, 2007); LHR (Gromoll et al, 2002;Martens et al, 2002;Huhtaniemi and Themmen, 2005;Piersma et al, 2007); FSHR (Rannikko et al, 2002;Meduri et al, 2003;Huhtaniemi and Themmen, 2005); TSHR (Biebermann et al, 1997;Costagliola et al, 1999;Tonacchera et al, 2000Tonacchera et al, , 2004Calebiro et al, 2005;Davies et al, 2005); E-BR (Tanaka et al, 1998;Fuchs et al, 2001;Chen et al, 2006;Tao, 2006); MC1R-4R (Beaumont et al, 2005;Clark et al, 2005;Govaerts et al, 2005;Tao, 2005Tao, , 2006Farooqi and O´Rahilly, 2006;Hinney et al, 2006;Lubrano-Berthelier et al, 2006;Alharbi et al, 2007;Lin et al, 2007); and CCR5 receptor (Lede...…”

Two Novel Transcripts for Human Endothelin B Receptor Produced by RNA Editing/Alternative Splicing from a Single Gene

“…Mutations in the ETB gene may lead to HSCR when ETB proteins are functionally altered. Among the reported ETB mutations, the mutation C109R resulted in a mutant ETB with a lowered affinity to ET-1, and the mutations W276C and S390R impaired intracellular signaling events (11). On the other hand, the ETB mutation N104I was found in a patient, but the protein exhibited normal functional properties, and this was designated a polymorphism (11).…”

“…Among the reported ETB mutations, the mutation C109R resulted in a mutant ETB with a lowered affinity to ET-1, and the mutations W276C and S390R impaired intracellular signaling events (11). On the other hand, the ETB mutation N104I was found in a patient, but the protein exhibited normal functional properties, and this was designated a polymorphism (11). In investigating the ETB gene in three patients with HSCR, we did not detect the mutations mentioned above, but mutant as well as full-length transcripts of the ETB gene were found in one patient (HD8).…”