2000 Global Researcher Conference Proceeding

March 10 - 12, 2000

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Conference: 2000 Global Researcher Conference
Title: Naturally occurring and in vitro mutations defining the role of the NPXXY motif in the vasopressin V2 receptor
Authors: Oksche, Alexander; Furkert, Jens; Krause, Gerd; Rosenthal, Walter
Institutions: Institut Fuer Pharmakologie, Forschungsinstitut fur Molekulare Pharmakologie, Forschunginstitut fur Molekulare Pharmakologie (FMP), Charite - Universitatsmedizin Berlin
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Oksche

G protein-coupled receptors represent one of the largest protein families in multicellular organisms. Among the identified GPCRs at least 3 groups, defined by distinct structural features, can be distinguished. According to its structural features the vasopressin V2 receptor belongs to the class I/rhodopsin-like receptors, as it displays a DRY motif (at the junction of the third transmembrane domain and the second intracellular loop) and an NPXXY motif (at the end of seventh transmembrane domain). The highly conserved NPXXY motif was found to be of variable importance for ligand binding and G protein activation in a number of class I receptors, e.g. ß2 adrenergic receptor, angiotensin II receptor. Its role in the function of the V2 receptor has not been established, but is of clinical interest as various disease-causing mutations have been found in the motif (e.g. P322S, P322H, W323R). We therefore analyzed the naturally occurring mutants P322S, P332H and W323R and other in vitro mutations in order to evaluate the possible role of P322, W323 and Y325 in receptor function. Expression of the V2 receptor mutants P322S, P322H and W323R in COS.M6 and HEK 293 cells revealed a loss of function. While the mutants P322H and W323R did not confer any [3H]AVP binding to or AVP-mediated accumulation of cAMP in intact cells, the P322S mutant receptor did cause AVP-sensitive accumulation of cAMP. However, we were unable to detect [3H]AVP binding to intact cells, suggesting a very low number of P322S mutant receptors present at the cell surface, which, though sufficient to stimulate the Gs/adenylyl cyclase system, was below the detection limit of the binding experiments. The data suggest that W323 is orientated towards the hydrophobic membrane and thus does not tolerate replacement by charged residues. In addition, P322 is more likely to have a structural function than to be crucial for receptor signalling.

Replacement of Y325 within the NPXXY motif by serine, alanine or phenylalanine does not alter the receptor´s affinity for [3H]AVP in saturation analysis of intact, stably expressing CHO clones. In displacement analyses with [3H]AVP as tracer and AVP or dDAVP as competing ligands intact cells expressing the wild-type or the mutant V2 receptors (Y325S, Y325A, Y325F) revealed variable affinities for AVP and dDAVP. The wild-type V2 receptor displays lower Ki values for AVP than for dDAVP (2.9 +/-1.2 nM and 22.5 +/- 6.5 nM respectively). Replacement of tyrosine residue 325 by serine increases the affinity for dDAVP without changing the affinity for AVP, whereas replacement by the hydrophobic phenylalanine residue in the Y325F mutant reduces the affinity for dDAVP. In addition, the amino acid replacements of tyrosine 325 result in a rightshift in the EC50 values for the AVP-mediated cAMP accumulation of intact cells (WT

A structural feature of the vasopressin V2 receptor (V2R) is the NPXXY motif. It is located in the end of a section of the V2R known as the seventh transmembrane domain. Several mutations of the V2R gene have occurred in this NPXXY motif: P322S, P322H, W323R. In the first two mutations, the amino acid proline (P; the 322nd amino acid residue of the V2R protein) is replaced by the amino acid, serine (S) or histidine (H). In the third mutation, the amino acid tryptophan (W) is replaced by arginine (R).

Oksche et al., analyzed these mutations in order to determine the possible role P322, W323 and Y325 play in V2R function. The researchers expressed the three mutants in laboratory cell cultures and found these mutants did not function like normal V2Rs. P322H and W323R could not bind the natural ligand arginine vasopressin (AVP), and failed to induce AVP-mediated accumulation of cAMP. The P322S mutant, however, could cause a small, but significant AVP-mediated accumulation of cAMP. In addition, P322S was barely transported to the cell membrane, which is vital if it is to function normally.

This research suggests that W323 cannot be replaced by charged amino acid residues if the V2R is to function normally. P322, they found, is more likely to be concerned with the proper structure of V2R than with its ability to signal. Y325 appears to be involved in G protein coupling/activation but is not critical to it. In addition, Y325 appears to help stabilize either the seventh transmembrane domain or the overall V2R structure.