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.

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.

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.

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.

The protein tyrosine kinase pp60c-src is activated upon platelet stimulation.

Stimulation of human platelets causes a dramatic increase in phosphorylation of various proteins at tyrosine residues. The abundance of protein tyrosine kinases of the src-family in platelets, particularly pp60c-src, suggests an important role of these kinases in response to stimulation events. We have shown that pp60c-src is activated on agonist-induced platelet stimulation with respect to its substrate affinity. This was accompanied by phosphorylation of pp60c-src at Ser-12, a residue which is phosphorylated by PKC. Inhibition of PKC with a specific inhibitor, Ro-31-8220, suppressed thrombin-induced translocation of pp60c-src to the cytoskeleton. On the basis of our data, we suggest that the cytoskeletal association of pp60c-src is dependent on phosphorylation of pp60c-src at Ser-12 by PKC. Phosphorylation at Ser-12 in the membrane-binding domain might be the signal that displaces pp60c-src from the plasma membrane and, accompanied with the increased substrate affinity, facilitates phosphorylation of putative substrates.

Heparin-associated thrombocytopenia: the effects of various intravenous IgG preparations on antibody mediated platelet activation--a possible new indication for high dose i.v. IgG.

The immunologic type of heparin-associated thrombocytopenia (HAT) is caused by antibodies which activate platelets via the Fc-receptor in the presence of polysulfated oligosaccharides. The antigen is formed by a releasable platelet protein (in many cases PF4) complexed to heparin. Since the role of GP IIb/IIIa in platelet activation by HAT antibodies is controversial, we investigated platelet activation by antibodies related to HAT. We used normal platelets and platelets from a patient with Glanzmann's thrombasthenia (GT) lacking GP IIb/IIIa. Heparin and sera from patients with HAT stimulated GT platelets in the same manner as determined by 14C-serotonin release and the changes in phosphorylation of p20 and p47. Platelet activation could be inhibited by an anti FcRII monoclonal antibody (IV. 3, Fab-fragments), and by Fc-fragments, but not by F(ab')2-fragments of human IgG. The effect of four different, commercially available preparations of intact i.v. IgG on the platelet activation by six HAT sera was investigated by 14C-serotonin release. The inhibitory effect was strongly dependent upon the manufacturing process. At a concentration of 20 mg/ml only IgG that had been subjected to low pH and traces of pepsin sufficiently inhibited platelet activation. IgG treated with polyethylenglycol or sulfitolysis was less effective, whereas beta-propiolactone-treated IgG almost completely lost the ability to inhibit platelet activation by antibodies related to HAT. We conclude that inhibition of GP IIb/IIIa-fibrinogen interaction is insufficient for preventing platelet activation in HAT.(ABSTRACT TRUNCATED AT 250 WORDS)

[Fc receptor-dependent platelet activation results independently of glycoprotein complex IIb/IIIa]. / Die Fc-Rezeptor-abhängige Thrombozytenaktivierung erfolgt unabhängig vom Glykoproteinkomplex IIb/IIIa.

Platelets of a patient with Glanzmann's thrombasthenia revealed the same activation pattern when stimulated with antibodies of patients with heparin-associated thrombocytopenia (HAT) or immune complexes. This was investigated by the 14C-serotonin release test and by changes in phosphorylation of p20 and p47. Platelet activation by HAT antibodies was completely inhibited by a moab against the platelet FcRII (IV. 3) and by Fc fragments of human IgG but not by F(ab)2 fragments. We conclude that platelet activation via the FcRII occurs independently of the glycoprotein complex IIb/IIIa. Therapeutical approaches targeting GP IIb/IIIa-fibrinogen interaction seem to be not appropriate in HAT.

Substrate affinity of the protein tyrosine kinase pp60c-src is increased on thrombin stimulation of human platelets.

Human blood platelets contain high levels of non-receptor protein tyrosine kinases of the Src family, particularly pp60c-src, suggesting an important role for these enzymes in platelet physiology. Indeed, in response to various agonists of platelet function, a number of proteins become phosphorylated at tyrosine residues. However, no enzymic activation of an Src-related tyrosine kinase has yet been shown in platelets. In searching for the kinase(s) responsible, we found that all agonists tested that directly or indirectly activate protein kinase C in platelets (phorbol 12-myristate, 13-acetate, thrombin, vasopressin, collagen, calcium ionophore A23187) increased the overall activity of pp60c-src determined by IgG phosphorylation in an immunocomplex assay in the presence of low ATP concentrations. On the other hand, elevation of cyclic AMP directly by forskolin or indirectly by prostaglandin E1, or elevation of cyclic GMP by sodium nitroprusside did not significantly affect the activity of the enzyme. To substantiate the differences in enzyme activity, we determined Km and Vmax, values of pp60c-src from resting and thrombin-stimulated platelets. Thrombin treatment increased substrate affinity of pp60c-src as indicated by a 2- to 3-fold decrease in the Km values for ATP and the exogenous protein substrate casein. Vmax. values were only slightly altered under the assay conditions used. To further rule out modifications of pp60c-src in phosphorylation as a probable cause of the changed substrate affinity, we analysed tryptic phosphopeptides of immunoprecipitated, 32P-labelled pp60c-src of unstimulated and stimulated platelets. The platelet agonists listed above induced an increase in pp60c-src phosphorylation at Ser-12, which is the amino acid phosphorylated by protein kinase C. Surprisingly, we found that elevation of cyclic AMP did not affect 32P labelling of pp60c-src. On the basis of our data, we suggest that phosphorylation at Ser-12 might be one of the signal-triggering events that cause the increase in substrate affinity of pp60c-src.

Heparin-associated thrombocytopenia: immune complexes are attached to the platelet membrane by the negative charge of highly sulphated oligosaccharides.

The interaction of sulphated oligosaccharides (SO) with platelets and the antibody of heparin-associated thrombocytopenia (HAT type II) was investigated. 3H-heparin binding to platelets was inhibited by different SO, depending on their grade of sulphation. Dextran sulphate, pentosan polysulphate, and heparin were more effective than were LMW heparins. De-N-sulphated heparin and a LMW heparinoid (Org 10172) had no effect. Platelets preincubated with high-grade SO and washed, released serotonin in the presence of HAT sera without additional heparin. Platelets preincubated with HAT sera and then washed were not activated when heparin was added. Only high-grade SO which inhibited heparin binding to platelets caused platelet activation with HAT sera. However, low- and high-grade SO in high concentrations (0.11 g/l) inhibited serotonin release induced by HAT sera and heparin. 32P-phosphorylation of platelet proteins was enhanced by HAT-IgG and heparin and by heat-aggregated IgG, and was inhibited by the moab IV.3. High SO concentrations inhibited only the effect of HAT-IgG and not that of aggregated IgG. We assume that the antigen in HAT involves a releasable platelet protein with a binding site for SO. This was corroborated by studies with an anti-platelet factor 4 antibody causing Fc-receptor dependent platelet activation inhibitable by high SO concentrations.