1999 European Regional Conference Proceeding
May 12 - 16, 1999
| Conference: | 1999 European Regional Conference |
|---|---|
| Title: | Analysis of naturally occurring and in vitro mutations of the V2 receptor gene interfering with proper splicing |
| Authors: | Oksche, Alexander; Kuhn, Ralph; Furkert, Jens; Rosenthal, Walter |
| Institutions: | Institut Fuer Pharmakologie, Forschungsinstitut fur Molekulare Pharmakologie, Charite - Universitatsmedizin Berlin, Rudolf-Buchheim-Institut fur Pharmakologie |
Nephrogenic diabetes insipidus is, in most cases, caused by inactivating mutations of the vasopressin V2 receptor gene. Identification of the exact molecular mechanisms leading to the disease is of great importance for gaining insight into structure-function relationships. This goal is usually achieved by introducing mutations into the cDNA by site-directed mutagenesis, followed by transfection of the mutated cDNAs into eukaryotic cells and subsequent functional analysis of the heterologously expressed proteins. However, mutations located at the exon/intron boundaries, which may interfere with splicing, cannot be analyzed by this approach. The V2 receptor is expressed almost exclusively in the renal collecting duct. Tissue derived from the kidney is the only known source to display V2 receptor transcripts, which is rarely available. We cloned a genomic fragment of the V2 receptor gene harboring the complete coding region including the two introns. The donor and acceptor splice sites of the second intron were mutated separately or in combination. The plasmids harboring the wild-type and the various mutated genomic fragments were expressed in CHO- and MDCK cells. Total RNA of these cells was isolated and analyzed by northern blotting and RT-PCR. Mutation of the donor and/or acceptor splice sites resulted in the retention of the second intron. In addition, the donor splice site mutation caused the skipping of the second exon and the acceptor splice site mutation resulted in the activation of a cryptic acceptor splice site. We also analyzed an NDI-causing mutation in which a single base deletion at the junction of intron 2 and exon 3 has been identified. Expression of this mutant in our system revealed that the mutation does not interfere with proper splicing. However, the single base deletion shifts the reading frame and codes for a V2 receptor with an altered seventh transmembrane domain and an altered and extended intracellular C-terminus.
Congenital nephrogenic diabetes insipidus is most commonly caused by mutations of the vasopressin-2 receptor (AVPR2) gene.
Genes are comprised of sections called exons, which contain the code for the protein that the gene helps to produce, and sections called introns, which contain no code. An AVPR2 gene contains three exons and two introns. When AVPR2 gene mutations occur at exon/intron junctions, they may interfere with the gene´s ability to splice, i.e. attach individual exonic DNA molecules to each other. Also, AVPR2 genes with mutations at an exon/intron boundary cannot be analyzed in the normal way.
Oksche, et al., analyzed such a mutation by cloning a genomic fragment of the AVPR2 gene containing all the exons and introns. The donor and acceptor splice sites of the second intron were mutated either separately or in combination, and the mutated AVPR2 genes were expressed in laboratory cell cultures.
By analyzing these expressed genes, the researchers found that mutation of the donor and/or acceptor splice sites resulted in the retention of the second intron. When only the donor splice site was mutated, it caused skipping of the second exon. When only the acceptor splice site was mutated, a previously hidden acceptor splice site was activated.
The group also analyzed an AVPR2 gene with a mutation at the junction of intron 2 and exon 3 (single base deletion). Though this mutation causes NDI, it does not interfere with splicing. It does, however, result in a vasopressin-2 receptor that has alterations in two of its parts, the seventh transmembrane domain, which is the last fold of the protein within the cell membrane, and the tail end of the protein, the intracellular C-terminus.