The RET proto-oncogene encodes a receptor tyrosine kinase required for development of the kidney and neural crest-derived cell types. Alternative splicing of the 3′ exons of human RET results in three protein isoforms with distinct C-termini: RET9, RET51, and RET43. These RET isoforms show differential binding to downstream adapter molecules, suggesting they may have distinct signaling functions. We have characterized Ret 3′ sequences in mouse and investigated alternative splicing of this region. We found that the organization of Ret 3′ sequences is very similar to human RET. The mouse locus also has alternatively spliced C-terminal coding regions, and the sequences corresponding to RET9 and RET51 are highly conserved in both position and sequence with the human locus. Further, we compared the predicted C-terminal amino acids of RET9 and RET51 in seven vertebrate species, and found that they are well conserved. We have identified sequence encoding a putative ret43 isoform in mouse, however the predicted amino acid sequence showed low homology to human RET43. Our data suggest that RET isoforms are evolutionarily highly conserved over a broad range of species, which may indicate that each isoform has a distinct role in normal RET function.

The glial-cell-line-derived neurotrophic factor (GDNF) family receptors alpha (GFRalpha) are cell surface bound glycoproteins that mediate interactions of the GDNF ligand family with the RET receptor. These interactions are crucial to the development of the kidney and some peripheral nerve lineages. In humans, mutations of RET or RET ligands are associated with the congenital abnormality Hirschsprung disease (HSCR) in which nerves and ganglia of the hind gut are absent. As the GFRalpha family are required for normal activation of the RET receptor, they are also candidates for a role in HSCR. The GFRA2 gene, which is required for the development of the myenteric nerve plexus, is an excellent candidate gene for HSCR. In this study, we cloned the human GFRA2 locus, characterized the gene structure, and compared it with other GFRA family members. We further investigated the GFRA2 gene for mutations in a panel of HSCR patients. GFRA2 has nine coding exons that are similar in size and organization to those of other GFRA family genes. We identified six sequence variants of GFRA2, four of which did not affect the amino acid sequence of the GFRalpha-2 protein. Two further changes that resulted in amino acid substitutions were found in exon 9 and were predicted to lie in the amino acid sequence encoding the glycosylphosphatidylinositol-linkage signal of GFRalpha-2. There was no difference in frequency of any of the sequence variants between control and HSCR populations. Our data indicate that members of the GFRA gene family are closely related in intron/exon structure and in sequence. We have not detected any correlation between sequence variants of GFRA2 and the HSCR phenotype.

Background-The GDNF family receptor alpha (GFR ) proteins are extracellular cell surface bound molecules that act as adapters in binding of the GDNF family of soluble neurotrophic factors to the RET receptor. These molecules are essential for development of many neural crest derived cell types and the kidney. Mutations in RET and in two members of the GDNF ligand family are associated with Hirschsprung disease (HSCR), a congenital absence of the enteric ganglia. Members of the GFR family are also candidates for HSCR mutations. One such gene is GFR -3, which is expressed in the peripheral nervous system and developing nerves. Objective-We have characterised the structure of the human GFR -3 locus and investigated the gene for sequence variants in a panel of HSCR patients. Methods-Long range PCR or subcloning of PAC clones was used to investigate GFR -3 intron-exon boundaries. A combination of single strand conformation polymorphism (SSCP) analysis and direct sequencing was used to investigate GFR -3 sequence variants. Results-GFR -3 spans eight coding exons and has a gene structure and organisation similar to that of GFR -1. We identified three polymorphic variants in GFR -3 in a normal control population, a subset of which also occurred in HSCR patients. We did not detect any sequence variants within the coding sequence of GFR -3. We found a base substitution in the 5' UTR of GFR -3, 15 base pairs upstream of the translation start site. A second substitution was identified in intron 4 (IVS4-30G>A) between the splice branch site and the splice acceptor site. The final variant was a 2 base pair insertion within the splice donor consensus sequence of exon 7 (IVS7+4ins GG). Conclusions-We did not detect any correlation between variants of GFR -3 and the HSCR phenotype. Our data suggest that mutations of this gene are not a cause of HSCR. (J Med Genet 2000;37:674-679)

Mutations of the RET proto-oncogene are found in the majority of patients with the inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN 2). A minority of cases, however, have no detectable RET mutation and there is considerable phenotypic variation within and among MEN 2 families with the same RET mutation, suggesting a role for other loci in this disease. A candidate for such a gene is glial cell line-derived neurotrophic factor receptor alpha 4 (GFRA4), which encodes a cell surfacebound co-receptor (GFRa4) required for interaction of RET with its ligand persephin. The GFRA4 gene has multiple alternative splices leading to three distinct protein isoforms that are prominently expressed in thyroid. We postulated that mutations of GFRA4 contribute to MEN 2 in the absence of RET mutations or modify the RET mutation phenotype. We screened patients with MEN 2 or MEN 2-like phenotypes, with and without RET mutations, for variants of GFRA4. We identified 10 variants, one of which was over represented in, and two of which were found exclusively in, our patient populations. One of these was a single-base substitution upstream of the GFRa4 coding region, where it may alter gene expression. The second was a 7 bp insertion, which results in a change in reading frame for all three GFRa4 isoforms. This would cause a relative shift in membrane bound and soluble forms of GFRa4, which would significantly alter the formation of RET signalling complexes. Our data suggest a model of wild-type GFRa4 isoform expression that includes both activating and inhibiting co-receptors for RET.

Collagen-binding heat-shock proteins of M(r) 46-47 kDa have been postulated to function as putative molecular chaperones in the biosynthesis of collagen in several species. The rat homologue of this family of heat-shock proteins is called gp46. In the present study, we employed Western blotting and immunohistochemical methods to determine the tissue distribution and cellular localization of gp46 in the thoracic aorta, heart, kidney, liver and lung of eight-day-old Wistar rats. Highest levels of gp46 were detected in the thoracic aorta and lung, followed by the kidney and heart. Gp46 levels were low to undetectable by Western blot analysis in the liver. Immunohistochemistry revealed that gp46 labelling was observed almost exclusively in three distinct cell types: fibroblasts, muscle cells, and some epithelial cells. Gp46 was detected in the fibroblasts of the hepatic triad, in the interstitium of the alveolar wall and in the tunica adventitia of blood vessels in the majority of tissues examined, in atrial and ventricular cardiomyocytes, in vascular smooth muscle cells of the abluminal portion of the tunica media, in parietal epithelial cells and mesangial cells of the glomerulus, in epithelial cells of the distal tubules and collecting ducts in the kidney and clusters of immature renal tubules, in epithelial cells of the bile duct, and in mesodermal cells surrounding the liver. These results demonstrate that gp46 is present in collagen producing cells and cells undergoing rapid growth and development, suggesting that gp46 may play a significant role in these processes.