1999 European Regional Conference Proceeding
May 12 - 16, 1999
| Conference: | 1999 European Regional Conference |
|---|---|
| Title: | Structure of V2 vasopressin receptor oligomers: evidence for contact dimer formation |
| Authors: | Schoneberg, Torsten; Schulz, Angela; Grosse, R.; Schultz, Prof. Dr. Med. Gunter; Gudermann, Thomas |
| Institutions: | University of Leipzig, Freie University of Berlin, Institut fur Pharmakologie |
We have recently shown that mutant vasopressin receptors (V2-Rs) harboring missense or nonsense mutations within the last third (TMD6-7) of the receptor molecule can be functionally rescued by supplying a receptor fragment spanning the mutated receptor portion. The targeted expression of specific receptor polypeptides may lead to novel strategies in the treatment of diseases such as nephrogenic diabetes insipidus caused by inactivating G-protein-coupled receptor (GPCR) mutations. The ability of receptor fragments to interact with different mutant V2-Rs is consistent with several recent reports suggesting that GPCRs can form dimers. Two structural models of dimer formation have been suggested. The contact interface of so called 'swapped dimers' is recruited from rearrangement of independent folding domains of individual receptor monomers. In 'contact dimers,' a lateral interaction of the individual receptor molecules is assumed. Functional complementation of two identical full-length receptors harboring different missense mutations in the N- and C-terminal domains would be the most convincing evidence demonstrating the existence of homodimers in a swapped domain structure. Taking advantage of receptor reconstitution from individual folding domains, we attempted to restore the function of missense mutations (R137H, S167L, R181C) within the N-terminal domain of the V2 vasopressin receptor (V2-R) by coexpressing mutated full-length (Y280C) and C-terminally truncated (E242X) receptors. Coimmunoprecipitation and ELISA studies demonstrated a specific association of C-terminally truncated receptors with the full-length V2-R. Despite this specific interaction, no functional proof of receptor-fragment association by a domain-swapped mechanism was achieved for intracellularly retained (R137H, S167L) or for properly transported (R181C) mutant V2-Rs. However, considerable functional activity of R137H and R181C upon coexpression with E242X was regained following mutational disruption of the extracellular disulfide bond which is highly conserved among GPCRs. In summary, we have shown that the V2-R forms oligomeric complexes with both full-length and truncated V2-R constructs. The lack of functional reconstitution of missense mutations upon coexpression with mutant receptors or N-terminal receptor fragments supports an oligomer structure by lateral interaction rather than by a domain swapped mechanism. The latter mechanism can only be achieved after major structural alteration of the folding domain interface
Schoneberg et al., demonstrated that mutant V2 vasopressin receptors (AVPR2s) rendered dysfunctional due to missense or nonsense mutations located in the last third of their structure could be made functional by supplying mutant AVPR2s or AVPR2 fragments that cover the portion of the mutant AVPR2 that is mutated. This ability of receptor fragments to interact with different mutant AVPR2s may provide a useful treatment methodology for NDI. A hypothetical model explaining this phenomenon is that AVPR2s can form dimers. Dimers are complex molecules formed by two or more identical, yet simpler AVPR2 molecules. The fine structure of such dimers are unknown. Some researchers think they do this by swapping dimers wherein independent sections of the AVPR2 called folding domains rearrange themselves with other independent folding domains. Other researchers hold another explanation of how the AVPR2 fragments come together to create a dimer complex. Their model is called contact dimers and assumes that the individual receptor molecules interact with each other laterally.
Schoneberg, et al., attempted to restore the function of three AVPR2s with missense mutations in the beginning section of their structure (the N-terminus) by coexpressing them with a full length, though still mutated, AVPR2 and an AVPR2 whose mutation left it missing its tail end (C-terminus). The coexpression produced a specific association of the mutant AVPR2 missing its C-terminus with the full length AVPR2. This interaction did not, however, restore functional ability to the three mutant AVPR2s with mutations in their N-terminus. However, two of these three mutant AVPR2s did exhibit functional activity upon coexpression with the AVPR2 missing its C-terminus after the researchers mutationally disrupted the AVPR2s extracellular disulfide bond.
This showed that the AVPR2 can form complexes with both full-length and truncated AVPR2 constructs, a phenomena which the researchers say occurs through lateral interaction rather than by swapping domains.