2004 Global Researcher Conference Proceeding
April 09 - 11, 2004
| Conference: | 2004 Global Researcher Conference |
|---|---|
| Title: | Characterization of mutant vasopressin V2 receptors with a misfolded AVP binding site |
| Authors: | Krause, Gerd; Wiesner, Burkhard; Wuller, Stefan; Schulein, Ralf; Rosenthal, Walter; Oksche, Alexander |
| Institutions: | Forschunginstitut fur Molekulare Pharmakologie (FMP), University Hospital of the RWTH Aachen, Forschungsinstitut fur Molekulare Pharmakologie (FMP), Charite - Universitatsmedizin Berlin, Institut Fuer Pharmakologie |
X-linked nephrogenic diabetes insipidus (NDI) is characterized by a resistance of the kidney to the action of AVP. In most cases NDI is caused by inactivating mutations of the vasopressin V2R receptor (V2R) gene. More than 150 different mutations have been described of which roughly 50 % encode proteins with the substitution of a single amino acid (missense mutations). Expression analysis revealed that a small number of mutant V2Rs reaches the plasma membrane, but lacks affinity to AVP and/or fails to stimulate the adenylyl cyclase system. The majority of V2R mutants is retained in the endoplasmic reticulum (ER) by the quality control system. For a variety of ER-retained mutants, however, cell surface expression can be restored by non-peptide antagonists. So far, binding-defective V2R mutants and mutants sensitive for antagonist-mediated restoration of cell surface expression are only poorly characterized.
Therefore, we tested mutant V2Rs either lacking AVP-binding (C192A, F287L) or undergoing antagonist-mediated cell surface expression (C319Y) for ligand-dependent differences in their ability to stimulate adenylyl cyclase. In HEK293 cells, transiently expressing the C192A and F287L mutant V2Rs almost no specific binding of [3H]AVP was found. Upon stimulation with AVP only cells expressing the F287L mutant revealed a small increase in cAMP levels (cAMP RIA). When we tested crude membrane preparations of HEK293 cells transiently expressing the mutant V2Rs for their ability to stimulate adenylyl cyclase in the presence of various AVP analogues (adenylyl cyclase assays), none of the agonists tested elicted a higher increase in cAMP formation than found for AVP.
The mutant C319Y, which is mainly retained in the ER, shows cell surface expression in the presence of SR49059 and SR121463B (V1AR- and V2R-selective antagonists, respectively). Quantitative analysis by laser scanning microscopy revealed that both SR49059 and SR121463B increased the expression of C319Y at the cell surface 5 to 6-fold. However, the increase in antagonist-mediated plasma membrane fluorescence was not accompanied by a comparable increase in [3H]AVP binding. Upon stimulation of intact cells expressing the C319Y mutant with AVP an increase in cAMP was observed, which was not significantly different from non-antagonist-treated controls. Similar results were obtained for the synthetic AVP analogues.
The data suggest that antagonist-mediated restoration of cell suface expression of the C319Y mutant V2R mainly results in receptors lacking significant affinity to AVP. In the case of the mutants C192A and F287L no or only a slight AVP-mediated stimulation of adenylyl cyclase was observed. In no case the synthetic ligands elicited a better stimulation of adenylyl cyclase than AVP. Thus, the mutants show complex defects of the ligand binding site, which are obviously not recognized by the quality control system.
Scientists have recorded over 150 mutations within the vasopressin 2 receptor (V2R) gene that result in X-linked NDI. A majority of these mutations result in mutant V2Rs that are retained in the endoplasmic reticulum (ER) and do not reach the cell membrane. A small number of mutant V2Rs do reach the cell membrane, but are unable to bind the antidiuretic hormone, arginine-vasopressin (AVP) and/or fail to stimulate the adenylyl cyclase system. Researchers have discovered that some mutant V2Rs that normally are retained in the ER can be guided to the cell membrane when they bind to membrane-permeable molecular structures called non-peptide antagonists (there are many types of antagonists and only those specific for the V2R can help to rescue mutant V2Rs.)
Oksche et al., tested two mutant V2Rs that are transported to the cell membrane, but are unable to bind to AVP. They wanted to see if there were any differences in the ability of the V2Rs to stimulate adenylyl cyclase. The researchers found that adding AVP or synthetic analogues of AVP to cells expressing the mutant V2Rs did not result in a significant stimulation of the adenylyl cyclase.
Further, the researchers investigated an ER-retained V2R mutant that is able to achieve cell membrane expression following binding to a specific vasopressin receptor antagonist. When the researchers incubated cells expressing the ER-retained V2R mutant with the vasopressin receptor antagonists, the mutant V2R reached the cell membrane in five to six times the numbers that it usually does. However, this did not increase its ability to bind to AVP. Similarly, AVP and a variety of synthetic analogues failed to stimulate the adenylyl cyclase.