1998 Global Conference Proceeding
March 02 - 04, 1998
| Conference: | 1998 Global Conference |
|---|---|
| Title: | Supplementation of Receptor Fragments: An approach to Functionally Rescue Mutant V2 Vasopressin Receptors |
| Authors: | Schoneberg, Torsten; Gudermann, Thomas; Wess, Jurgen; Schulz, Angela |
| Institutions: | University of Leipzig, Institut fur Pharmakologie, National Institutes of Health, NIDDK |
The structure of all G-protein-coupled receptors (GPCRs) is predicted to share a common molecular architecture characterized by the presence of seven x-helically arranged transmembrane domains (TM1-7) connected by three extracellular and three intracellular loops. Recent in vivo studies suggest that GPCRs are assembled from multiple independent folding units. Investigations with m2 and m3 muscarinic acetylcholine receptors, for example, have shown that co-expression of receptor fragments resulting from ‘splitting’ these receptors in various intracellular and extracellular loops leads to the appearance of functional receptor proteins. Individually expressed receptor fragments were found to be localized at the plasma membrane in correct orientation underpinning their independent folding unit character. These finding prompted us to apply this strategy to restore the function of clinically relevant GPCRs harboring inactivating mutations. X-linked nephrogenic diabetes insipidus (NDI) is a disorder characterized by the excretion of abnormally large volumes of diluted urine due to mutations in the V2 vasopressin receptor (V2-R) gene. The vast majority of mutations found in the V2-R gene are single amino acid substitutions (missense mutations) but more than one third of all V2-R mutations, mainly nonsense or frameshift mutations, result in a premature stop of receptor protein translation. To test whether inactivating mutations could be rescued by co-expression of receptor fragments, a series of mutant V2-Rs known to be responsible for NDI were used as a model system. Several mutant receptors regained considerable functional activity in COS-7 cells upon co-expression with a C-terminal V2-R polypeptide spanning the sequence where the various mutations occur. Using a co-immunoprecipitation as well as a sandwich ELISA strategy, we demonstrated that the C-terminal V2-R fragment can associate directly with the mutant V2-Rs in a specific manner. Subsequently, we tested the potential therapeutic usefulness of this co-expression strategy. Toward this aim, CHO cell lines expressing low levels of functionally inactive V2-Rs which more closely mimic the physiological situation were infected with a recombinant adenovirus carrying the C-terminal V2-R fragment. Whereas non-infected CHO cells expressing mutant V2-Rs did not show a function upon addition of arginine vasopressin (AVP), infection with recombinant adenovirus enabled AVP to induce a pronounced cAMP response.
In summary, our data suggest that adenoviral-mediated expression of receptor fragments results in a cell-specific molecular correction of functional V2-R defects and may lead to novel strategies in the treatment of NDI and other diseases caused by inactivating mutations in GPCRs.