Identification of Rab3-, Rab5a- and synaptobrevin II-like proteins in a preparation of rat kidney vesicles containing the vasopressin-regulated water channel

According to the 'shuttle hypothesis', vasopressin increases the water permeability of renal epithelial cells by exocytotic fusion of vesicles containing the water channel AQP-CD with the apical plasma membrane, whereas withdrawal of vasopressin results in endocytotic uptake of AQP-CD. The proteins involved in the redistribution of AQP-CD have not been identified. With a panel of monoclonal antibodies, we detected Rab3-, Rab5a- and synaptobrevin II-like proteins in a kidney preparation enriched in AQP-CD-containing vesicles. The synaptobrevin II-like proteins is not identical with the ubiquitous cellubrevin. Rab3- and synaptobrevin II- but not Rab5a-like proteins were co-enriched with AQP-CD. The data suggest that the proteins involved in hormonal regulation of water permeability in kidney epithelial cells are identical or similar to those involved in regulated exocytosis in secretory cells

Properties of the human arginine vasopressin V2 receptor after site-directed mutagenesis of its putative palmitoylation site.

Most G-protein-coupled receptors have conserved cysteine residues in their C-terminal cytoplasmic domain that appear to be generally palmitoylated. An example is the human arginine vasopressin V2 receptor with cysteine residues at positions 341 and 342. Site-directed mutagenesis of the putative palmitoylation site was used to study the significance of palmitoylation for the V2 receptor. A multifunctional expression plasmid was constructed by cloning the V2 receptor cDNA into the vector pCDNAI.Neo. The resulting plasmid allowed site-directed mutagenesis experiments without subcloning, and stable and transient expression of the V2 receptor in Ltk- and COS.M6 cells respectively. The conserved cysteine residues Cys-341 and Cys-342 were placed by serine residues, yielding the single mutants C-341S and C-342S and the double mutant C-341S/C-342S. Functional expression in stably transfected Ltk- cells showed that the affinity of the three mutant receptors for arginine vasopressin was not altered. In contrast with the activation of adenylate cyclase through beta 2 adrenergic receptors, arginine vasopressin stimulated adenylate cyclase to the same extent and with similar EC50 values in both wild-type and mutant receptors. Transient expression of the C-341S/C-342S mutant receptor in COS.M6 cells confirmed an unaltered affinity of the mutant receptor for arginine vasopressin. However, the number of arginine vasopressin-binding sites on the cell surface was reduced by 30%, suggesting that the transport of the mutant receptor to the cell surface was impaired. In addition, the decrease in detectable arginine vasopressin-binding sites on the cell surface following pre-exposure to hormone was reduced, indicating that the sequestration/internalization of the mutant receptor on the cell surface was affected. The present data indicate that palmitoylation of the V2 receptor is important for intracellular trafficking and/or sequestration/internalization but not for agonist binding or activation of the Gs/adenylate cyclase system

Vasopressin V2 receptor mutants that cause X-linked nephrogenic diabetes insipidus: analysis of expression, processing, and function

We investigated the biochemical and functional properties of five vasopressin V2 receptor mutants (L44F, L44P, W164S, S167L, and S167T) that were recently described in families with a history of X-linked nephrogenic diabetes insipidus. COS.M6 cells transfected with cDNA encoding these mutants acquired < 4% specific [3H]arginine vasopressin (AVP) binding sites on the cell surface in comparison with cells transfected with cDNA coding for the wild-type receptor. Membrane preparations from COS.M6 cells or human embryonic kidney 293 cells expressing these mutants did not respond with an increase in adenylyl cyclase activity in response to AVP, which is in contrast to membranes from cells expressing the wild-type. By analyzing fusion proteins of the V2 receptor and Escherichia coli alkaline phosphatase attached to the carboxyl terminus of the receptor moiety, we found that the mutants L44P, W164S, S167L, and S167T lacked complex glycosylation and were expressed at low levels. The data suggest that the mutants L44P, W164S, S167T, and S167L are misfolded and therefore retained within the endoplasmic reticulum and degraded. In contrast, the fusion proteins carrying the mutant L44F and the in vitro mutant S167A were expressed in their mature form at wild-type levels; however, only the mutant S167A was functionally active. Site-directed mutagenesis of S167 revealed that elimination of the endogenous hydroxyl group (S167A) yielded a protein with properties identical to those of the wild-type receptor, whereas both the introduction of a methyl group (S167T) and the replacement of the hydroxyl group by an isopropyl group (S167L) profoundly disturbed receptor processing. The data show that minute changes at codon 167 nearly abolish expression of a mature protein, thus defining structural requirements of this codon

Polarized expression of the vasopressin V2 receptor in Madin-Darby canine kidney cells

BACKGROUND: The vasopressin V2 receptor is expressed in the polarized principal cell of the renal collecting duct. Inactivating mutations of the vasopressin V2 receptor gene cause X-linked nephrogenic diabetes insipidus (NDI). Most of the mutant V2 receptors show transport defects, as analyzed in non-polarized cells, but data pertaining to polarized cells have not previously been presented. METHODS: Madin-Darby canine kidney cell (MDCK) II clones stably expressing c-myc-tagged human V2 receptors were characterized for [3H]-arginine vasopressin (AVP)-binding and AVP-sensitive adenylyl cyclase activity. The V2 receptors were immunocytochemically localized using the tyramide signal amplification technique in conjunction with an anti-c-myc antibody. RESULTS: The introduction of the c-myc epitope at the N- or C-terminus did not affect the functional properties of the V2 receptor expressed in MDCK II clones. However, the use of standard immunofluorescence methodology for these MDCK II clones yielded only weak signals. With the tyramide signal amplification technique, strong signals were obtained, showing the V2 receptor to be mainly localized within the lateral and, to a minor extent, apical membrane. In MDCK II clones stably expressing the c-myc-tagged V2 receptor NDI mutant L44P, fluorescent signals were found exclusively within the cell. CONCLUSION: The wild-type V2 receptor is expressed mainly in the lateral membrane, whereas the L44P mutant is completely retained within the cell. In conjunction with tyramide signal amplification, MDCK II cells constitute a suitable model for the analysis of transport-defective mutants of the V2 receptor

A heterotrimeric G protein of the Gi family is required for cAMP-triggered trafficking of aquaporin 2 in kidney epithelial cells

Vasopressin is the key regulator of water homeostasis in vertebrates. Central to its antidiuretic action in mammals is the redistribution of the water channel aquaporin 2 (AQP2) from intracellular vesicles to the apical membrane of kidney epithelial cells, an event initiated by an increase in cAMP and activation of protein kinase A. The subsequent steps of the signaling cascade are not known. To identify proteins involved in the AQP2 shuttle we exploited a recently developed cell line (CD8) derived from the rabbit cortical collecting duct and stably transfected with rat AQP2 cDNA. Treatment of CD8 cells with pertussis toxin (PTX) inhibited both the vasopressin-induced increase in water permeability and the redistribution of AQP2 from an intracellular compartment to the apical membrane. ADP-ribosylation studies revealed the presence of at least two major PTX substrates. Correspondingly, two alpha subunits of PTX-sensitive G proteins, Galphai2 and Galphai3, were identified by Western blotting. Introduction of a synthetic peptide corresponding to the C terminus of the Gi3 alpha subunit into permeabilized CD8 cells efficiently inhibited the cAMP-induced AQP2 translocation; a peptide corresponding to the alpha subunits of Gi1/2 was much less potent. Thus a member of the Gi family, most likely Gi3, is involved in the cAMP-triggered targeting of AQP2-bearing vesicles to the apical membrane of kidney epithelial cells