1998 Global Conference Proceeding
March 02 - 04, 1998
| Conference: | 1998 Global Conference |
|---|---|
| Title: | Structure-Function Analysis of the V2 Vasopressin Receptor |
| Authors: | Zhu, Xiangyang; Erlenbach, Isolde; Wess, Jurgen |
| Institutions: | National Institutes of Health, NIDDK, National Institutes of Health |
The molecular mechanisms governing V2 vasopressin receptor/Gs coupling selectivity and V2 receptor assembly were studied by a combined molecular genetic/biochemical approach. Previous studies with hybrid V1a/V2 vasopressin receptors showed that the Gs-coupling selectivity of the V2 receptor is largely determined by its third intracellular loop (i3) (Liu and Wess, J. Biol. Chem., 271, 8772, 1996). To identify specific subdomains or residues within this region that are of particular importance for proper Gs recognition, we systematically replaced distinct i3 loop segments/single amino acids in the V1a receptor with the corresponding V2 receptor sequences (residues) and studied whether the resulting hybrid receptors gained the ability to mediate hormone-dependent cAMP production. Functional analysis of these mutant receptors in transfected COS-7 cells indicated that the presence of V2 sequence at the N-terminal portion of the i3 loop is critical for efficient activation of Gs. More detailed mutational analysis of this receptor region showed that two polar V2 receptor residues, Gln225 and Glu231, play key roles in Gs recognition. Interestingly, preliminary data indicated that the efficiency of Gs coupling is also modulated by the length of the central portion of the i3 loop (rather than the specific amino acid sequence within this i3 subdomain).
We have previously shown that mutant V2 vasopressin receptors containing mutations in the C-terminal third of the receptor protein can be functionally rescued by coexpression with a C-terminal V2 receptor fragment spanning the region where the various mutations occur (Schöneberg et al., EMBO J., 15, 1283, 1996). In the present study, we have systematically analyzed the ability of N- and C-terminal V2 receptor fragments to interfere with the activity of the wild type V2 receptor expressed in COS-7 cells. Several N-terminal V2 receptor fragments were identified which strongly inhibited the function of the coexpressed full-length receptor, as determined in cAMP and radioligand binding assays. However, these truncation mutants did not interfere with the function of other Gs-coupled receptors such as the D1 dopamine and the ß2-adrenergic receptors. ELISA studies indicated that the N-terminal V2 receptor fragments, though poorly expressed on the cell surface themselves, strongly impaired cell surface trafficking of the wild type V2 receptor. In addition, Western analysis showed that all V2 receptor truncation mutants displaying dominant negative activity were able to form heterodimers with the full-length V2 receptor. These findings are consistent with the concept that G protein-coupled receptors can form oligomeric arrays and improperly folded mutant receptors can interfere with the proper function and intracellular trafficking of such complexes.
The V2 vasopressin receptor selectively interacts with the stimulatory G protein, Gs, resulting in the increase in intracellular cAMP levels. To identify regions in the V2 receptor that are critical for proper Gs recognition, Zhu et al., substituted different intracellular segments of the V2 receptor into the V1a vasopressin receptor, which is unable to couple to Gs. Through biochemical analysis of these hybrid receptors, the authors showed that two short intracellular segments of the V2 receptor are of critical importance for selective coupling to Gs.
Mutations in the V2 receptor can prevent it from functioning properly. Interestingly, the authors previously showed (in cultured cells) that V2 receptor fragments from a functional V2 can restore activity to selected mutant V2 receptors. In addition, Zhu et al., recently found that certain mutant V2 receptors can inhibit the function of the wild-type (full length) V2 receptor. They showed that this effect is most likely due to an intracellular complex formation between mutant and wild-type receptor which prevents the wild-type receptor from reaching the cell surface. These findings suggest that the V2 receptor, like other G protein-coupled receptors, can form aggregates, and that misfolded mutant receptors can interfere with the proper function of such complexes.