All silicon infrared photodiodes: photo response and effects of processing temperature.

CMOS compatible infrared waveguide Si photodiodes are made responsive from 1100 to 1750 nm by Si(+) implantation and annealing. This article compares diodes fabricated using two annealing temperatures, 300 and 475 degrees C. 0.25-mm-long diodes annealed to 300 degrees C have a response to 1539 nm radiation of 0.1 A W-(-1) at a reverse bias of 5 V and 1.2 A W(-1) at 20 V. 3-mm-long diodes processed to 475 degrees C exhibited two states, L1 and L2, with photo responses of 0.3 +/-0.1 A W(-1) at 5 V and 0.7 +/-0.2 A W(-1) at 20 V for the L1 state and 0.5 +/-0.2 A W(-1) at 5 V and 4 to 20 A W(-1)-1 at 20 V for the L2 state. The diodes can be switched between L1 and L2. The bandwidths vary from 10 to 20 GHz. These diodes will generate electrical power from the incident radiation with efficiencies from 4 to 10 %.

The early stages of the intracellular transport of membrane proteins: clinical and pharmacological implications.

Intracellular transport mechanisms ensure that integral membrane proteins are delivered to their correct subcellular compartments. Efficient intracellular transport is a prerequisite for the establishment of both cell architecture and function. In the past decade, transport processes of proteins have also drawn the attention of clinicians and pharmacologists since many diseases have been shown to be caused by transport-deficient proteins. Membrane proteins residing within the plasma membrane are transported via the secretory (exocytotic) pathway. The general transport routes of the secretory pathway are well established. The transport of membrane proteins starts with their integration into the ER membrane. The ribosomes synthesizing membrane proteins are targeted to the ER membrane, and the nascent chains are co-translationally integrated into the bilayer, i.e., they are inserted while their synthesis is in progress. During ER insertion, the orientation (topology) of the proteins in the membrane is determined. Proteins are folded, and their folding state is checked by a quality control system that allows only correctly folded forms to leave the ER. Misfolded or incompletely folded forms are retained, transported back to the cytosol and finally subjected to proteolysis. Correctly folded proteins are transported in the membranes of vesicles through the ER/Golgi intermediate compartment (ERGIC) and the individual compartments of the Golgi apparatus ( cis, medial, trans) to the plasma membrane. In this review, the current knowledge of the first stages of the intracellular trafficking of membrane proteins will be summarized. This "early secretory pathway" includes the processes of ER insertion, topology determination, folding, quality control and the transport to the Golgi apparatus. Mutations in the genes of membrane proteins frequently lead to misfolded forms that are recognized and retained by the quality control system. Such mutations may cause inherited diseases like cystic fibrosis or retinitis pigmentosa. In the second part of this review, the clinical implications of the early secretory pathway will be discussed. Finally, new pharmacological strategies to rescue misfolded and transport-defective membrane proteins will be outlined.

Sorting functions of the individual cytoplasmic domains of the G protein-coupled vasopressin V(2) receptor in Madin Darby canine kidney epithelial cells.

Previous studies have shown that the G protein-coupled human vasopressin V(2) receptor (V(2) receptor) is expressed predominantly in the basolateral membrane of Madin Darby canine kidney type II (MDCKII) epithelial cells at steady state. Here we have assessed the influence of the individual cytoplasmic domains of the V(2) receptor on polarized sorting in MDCKII cells. The second (ICL2) and third (ICL3) intracellular loops and the C-terminal tail were fused separately to a green fluorescent protein-tagged receptor fragment comprising the first transmembrane domain and flanking regions. We show that the ICL2 domain of the V(2) receptor alone promotes basolateral cell surface expression and thus seems to contain the basolateral sorting signal of the V(2) receptor. Fusion of the other cytoplasmic domains, however, does not lead to a randomized cell surface expression. The C-terminal tail of the V(2) receptor promotes apical targeting. Fusion of ICL3 leads to a receptor fragment that is retained in the endoplasmic reticulum (ER). The results are consistent with a model in which the V(2) receptor contains signals for both apical and basolateral cell surface expression, the latter being dominant. Furthermore, ICL3 may contain a RXR [corrected] ER retention signal, which is not accessible in the correctly folded full-length receptor but which is unmasked when ICL3 is fused alone.

Cutting edge: antibody-mediated cessation of hemotropic infection by the intraerythrocytic mouse pathogen Bartonella grahamii.

The genus Bartonella includes important human-specific and zoonotic pathogens which cause intraerythrocytic bacteremia in their mammalian reservoir host(s). It is accepted that cellular immunity plays a decisive role in the host's defense against most intracellular bacteria. Bartonella sp. infection in the immunocompetent host typically leads to immunity against homologous challenge. The basis of this immunity, be it cellular or humoral, is unclear. In this study, the course of Bartonella grahamii bacteremia in immunocompetent and immunocompromised mice was compared. In immunocompetent hosts, the bacteremia is transient and induces a strong humoral immune response. In contrast, bacteremia persists in immunocompromised B and T cell-deficient mice. Immune serum transfer beginning with day 6 postinfection to B cell-deficient mice unable to produce Igs converted the persistent bacteremia to a transient course indistinguishable from that of immunocompetent animals. These data demonstrate an essential role for specific Abs in abrogating the intraerythrocytic bacteremia of B. grahamii in mice.

Invasion and persistent intracellular colonization of erythrocytes. A unique parasitic strategy of the emerging pathogen Bartonella.

The expanding genus Bartonella includes zoonotic and human-specific pathogens that can cause a wide range of clinical manifestations. A productive infection allowing bacterial transmission by blood-sucking arthropods is marked by an intraerythrocytic bacteremia that occurs exclusively in specific human or animal reservoir hosts. Incidental human infection by animal-adapted bartonellae can cause disease without evidence for erythrocyte parasitism. A better understanding of the intraerythrocytic lifestyle of bartonellae may permit the design of strategies to control the reservoir and transmittable stages of these emerging pathogens. We have dissected the process of Bartonella erythrocyte parasitism in experimentally infected animals using a novel approach for tracking blood infections based on flow cytometric quantification of green fluorescent protein-expressing bacteria during their interaction with in vivo-biotinylated erythrocytes. Bacteremia onset occurs several days after inoculation by a synchronous wave of bacterial invasion into mature erythrocytes. Intracellular bacteria replicate until reaching a stagnant number, which is sustained for the remaining life span of the infected erythrocyte. The initial wave of erythrocyte infection is followed by reinfection waves occurring at intervals of several days. Our findings unravel a unique bacterial persistence strategy adapted to a nonhemolytic intracellular colonization of erythrocytes that preserves the pathogen for efficient transmission by blood-sucking arthropods.

Functional rescue of the nephrogenic diabetes insipidus-causing vasopressin V2 receptor mutants G185C and R202C by a second site suppressor mutation.

Mutations in the gene of the G protein-coupled vasopressin V2 receptor (V2 receptor) cause X-linked nephrogenic diabetes insipidus (NDI). Most of the missense mutations on the extracellular face of the receptor introduce additional cysteine residues. Several groups have proposed that these residues might disrupt the conserved disulfide bond of the V2 receptor. To test this hypothesis, we first calculated a structure model of the extracellular receptor domains. The model suggests that the additional cysteine residues may form a second disulfide bond with the free, nonconserved extracellular cysteine residue Cys-195 rather than impairing the conserved bond. To address this question experimentally, we used the NDI-causing mutant receptors G185C and R202C. Their Cys-195 residues were replaced by alanine to eliminate the hypothetical second disulfide bonds. This second site mutation led to functional rescue of both NDI-causing mutant receptors, strongly suggesting that the second disulfide bonds are indeed formed. Furthermore we show that residue Cys-195, which is sensitive to "additional cysteine" mutations, is not conserved among the V2 receptors of other species and that the presence of an uneven number of extracellular cysteine residues, as in the human V2 receptor, is rare among class I G protein-coupled receptors.

Molecular and conformational features of a transport-relevant domain in the C-terminal tail of the vasopressin V(2) receptor.

We have previously shown a conserved glutamate/dileucine motif ((335)ELRSLL(340)) in the intracellular C terminus of the vasopressin V(2) receptor (V(2) receptor) to be essential for receptor transport from the endoplasmic reticulum (ER) to the Golgi apparatus. The motif may represent a transport signal that is recognized by a component of ER to Golgi vesicles. Alternatively, it may be necessary for transport-competent receptor folding to pass the quality-control system of the ER. To assess these two possibilities, we constructed a receptor fragment that allows transport studies independent of full-length receptor folding. Transmembrane domains II-VII were deleted, thereby fusing the intracellular C terminus to the first cytoplasmic loop. The mutations that impaired transport of the full-length receptor were introduced, and receptor fragments were localized in transiently transfected HEK 293 cells. All mutant receptor fragments were detectable at the plasma membrane, demonstrating that the glutamate/dileucine motif does not function as a small, linear vesicular transport signal. Instead, our data strongly suggest that this motif is required for transport-competent folding of the full-length receptor. To assess the underlying conformational features, a three-dimensional homology model of the V(2) receptor was computed. Our model predicts that the glutamate/dileucine motif contributes to a U-like loop within the intracellular C terminus. Residue Leu(339) may be required for folding back the intracellular C terminus to residue Leu(62) of the first cytoplasmic loop. We characterized the naturally occurring L62P and DeltaL62-R64 mutations in the first cytoplasmic loop and show that they lead to transport-defective full-length V(2) receptors that are retained in the ER, consistent with the structure model.

The role of conserved extracellular cysteine residues in vasopressin V2 receptor function and properties of two naturally occurring mutant receptors with additional extracellular cysteine residues.

The G protein-coupled vasopressin V2 receptor (V2 receptor) contains a pair of conserved cysteine residues (C112 and C192) which are thought to form a disulfide bond between the first and second extracellular loops. The conserved cysteine residues were found to be important for the correct formation of the ligand binding domain of some G protein-coupled receptors. Here we have assessed the properties of the V2 receptor after site-directed mutagenesis of its conserved cysteine residues in transiently transfected human embryonic kidney (HEK 293) cells. Mutant receptors (C112S, C112A and C192S, C192A) were non-functional and located mostly in the cell's interior. The conserved cysteine residues of the V2 receptor are thus not only important for the structure of the ligand binding domain but also for efficient intracellular receptor transport. In addition to the functional significance of the conserved cysteine residues, we have also analyzed the defects of two mutant V2 receptors which cause X-linked nephrogenic diabetes insipidus (NDI) by the introduction of additional cysteine residues into the second extracellular loop (mutants G185C, R202C). These mutations are assumed to impair normal disulfide bond formation. Mutant receptor G185C and R202C were efficiently transported to the plasma membrane but were defective in ligand binding. Only in the case of the mutant receptor R202C, the more sensitive adenylyl cyclase activity assay revealed vasopressin-stimulated cAMP formation with a 35-fold increased EC(50) value and with a reduced EC(max), indicating that ligand binding is not completely abolished. Taking the unaffected intracellular transport of both NDI-causing mutant receptors into account, our results indicate that the observed impairment of ligand binding by the additional cysteine residues is not due to the prevention of disulfide bond formation between the conserved cysteine residues.

A single negatively charged residue affects the orientation of a membrane protein in the inner membrane of Escherichia coli only when it is located adjacent to a transmembrane domain.

The orientation of membrane proteins is determined by the asymmetric distribution of charged residues in the sequences flanking the transmembrane domains. For the inner membrane of Escherichia coli, numerous studies have shown that an excess of positively charged residues defines a cytoplasmic domain of a membrane protein ("positive inside" rule). The role of negatively charged residues in establishing membrane protein topology, however, is not completely understood. To investigate the influence of negatively charged residues on this process in detail, we have constructed a single spanning chimeric receptor fragment comprising the N terminus and first transmembrane domain of the heptahelical G protein-coupled vasopressin V(2) receptor and the first cytoplasmic loop of the beta(2)-adrenergic receptor. When fused to alkaline phosphatase (PhoA), the receptor fragment inserted into the inner membrane of E. coli with its N terminus facing the cytoplasm (N(in)-C(out) orientation), although both membrane-flanking domains had rather similar topogenic determinants. The orientation of the receptor fragment was changed after the introduction of single glutamate residues into the N terminus. Orientation inversion, however, was found to be dependent on the location of the glutamate substitutions, which had to lie within a narrow window up to 6 residues distant from the transmembrane domain. These results demonstrate that a single negatively charged residue can play an active role as a topogenic determinant of membrane proteins in the inner membrane of E. coli, but only if it is located adjacent to a transmembrane domain.

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.

Prevention of acute allograft rejection by antibody targeting of TIRC7, a novel T cell membrane protein.

A novel 75 kDa membrane protein, TIRC7, is described that exhibits a central role in T cell activation in vitro and in vivo. Modulation of TIRC7-mediated signals with specific anti-TIRC7 antibodies in vitro efficiently prevents human T cell proliferation and IL-2 secretion. Moreover, anti-TIRC7 antibodies specifically inhibit type 1 subset specific IFN-gamma expression but spare the type 2 cytokine IL-4. Diminished proliferation but not IFN-gamma secretion is reversible by exogenous rIL-2. An anti-TIRC7 antibody that cross-reacts with the 75 kDa rat homolog exhibits inhibition of rat alloimmune response in vitro and significantly prolongs kidney allograft survival in vivo. Targeting of TIRC7 may provide a novel therapeutic approach for modulation of the immune response.

A dileucine sequence and an upstream glutamate residue in the intracellular carboxyl terminus of the vasopressin V2 receptor are essential for cell surface transport in COS.M6 cells.

Little is known concerning the intracellular transport of the G protein-coupled receptors (GPCRs). Previous studies suggested a functional role for those residues immediately preceding the conserved palmitoylated cysteine residues in the intracellular carboxyl termini of some GPCRs in cell surface transport. For the human vasopressin V2 receptor, we assessed the significance of a dileucine sequence with an upstream glutamate residue (ELRSLLCC) in mediating cell surface delivery. A series of deletion and point mutants in this region were constructed, and the mutant receptors were expressed in transiently transfected COS.M6 cells. By using [3H]arginine vasopressin binding assays to intact cells and immunofluorescence studies with intact and permeabilized cells, we show that residues E335 (mutant E335Q) and L339 (mutant L339T) are obligatory for receptor transport to the plasma membrane. Residue L340 has a minor but significant influence. [3H]Arginine vasopressin binding experiments on membranes of lysed cells failed to detect any intracellular binding sites for the transport-deficient mutant receptors, suggesting that residues E335 and L339 participate in receptor folding. Studies with green fluorescent protein-tagged receptors demonstrate that the bulk of the mutant receptors E335Q and L339T are trapped in the endoplasmic reticulum. Complex glycosylation was absent in these mutant receptors, supporting this conclusion. These data demonstrate that the glutamate/dileucine motif of the vasopressin V2 receptor is critical for the escape of the receptor from the endoplasmic reticulum, most presumably by establishing a functional and transport-competent folding state. A databank analysis revealed that these residues are part of a conserved region in the GPCR family.

Folding and cell surface expression of the vasopressin V2 receptor: requirement of the intracellular C-terminus.

We characterized truncations of the human vasopressin V2 receptor to determine the role of the intracellular C-terminus (comprising about 44 amino acids) in receptor function and cell surface expression. In contrast to the wild-type receptor, the naturally occurring mutant R337X failed to confer specific [3H]AVP binding to transfected cells. In addition, no vasopressin-sensitive adenylyl cyclase was detectable in membrane preparations of these cells. Laser scanning microscopy revealed that c-myc epitope- or green fluorescent protein-tagged R337X mutant receptors were retained within the endoplasmic reticulum. Increasing the number of C-terminal residues (truncations after codons 348, 354 and 356) restored G protein coupling, but revealed a length-dependent reduction of cell surface expression. Replacement of positively charged residues within the C-terminus by glutamine residues also decreased cell surface expression. A chimeric V2 receptor with the C-terminus replaced by that of the beta2-adrenergic receptor did not bind [3H]AVP and was retained within the cell. These data suggest that residues in the N-terminal part of the C-terminus are necessary for correct folding and that C-terminal residues are important for efficient cell surface expression.

Polarized cell surface expression of the green fluorescent protein-tagged vasopressin V2 receptor in Madin Darby canine kidney cells.

We have analyzed the polarized cell surface expression of the G protein-coupled vasopressin V2 receptor (V2 receptor) in Madin-Darby canine kidney (MDCK) epithelial cells by both conventional cell surface biotinylation assays and laser scanning microscopy of green fluorescent protein (GFP)-tagged receptors. Cell surface biotinylation assays with stably transfected filter-grown cells expressing alkaline phosphatase (PhoA)-tagged receptors demonstrated that the V2 receptor is located predominantly basolaterally at steady state, while minor amounts are expressed apically. Laser scanning microscopy of filter- and glass-grown MDCK cells stably transfected with a GFP-tagged V2 receptor confirmed that the receptor is expressed mainly basolaterally; within the basolateral compartment, however, the receptor was confined to the lateral subdomain. The results obtained with the GFP-tagged receptor are thus consistent with and refine those from the biotinylation assay, which does not discriminate lateral from basal membrane regions. Our data indicate that the GFP methodology may effectively supplement cell surface biotinylation assays in future studies of polarized receptor transport. We finally show that microinjection of a plasmid encoding the GFP-tagged V2 receptor into the nucleus of MDCK cells led to the same results as experiments with stably transfected cells. However, since there was no need for selecting stably transfected cell lines, the experiments were complete within hours. The microinjection technique thus constitutes a powerful single cell technique to study the intracellular transport of G protein-coupled receptors. The methodology may be applicable to any cell type, even to tissue-derived, primary cultured cells; coinjection of transport-regulating compounds should also be possible.

Topology of eukaryotic multispanning transmembrane proteins: use of LacZ fusions for the localization of cytoplasmic domains in COS.M6 cells.

In Escherichia coli, the topology of inner membrane proteins can be studied conveniently with the alkaline phosphatase/beta-galactosidase (PhoA/LacZ) gene fusion system. PhoA is enzymatically active only when fused to external domains, LacZ when fused to cytoplasmic domains. In eukaryotic cells, only time consuming methods exist to study the topology of membrane proteins. We have extended in the first systematic study the PhoA/LacZ gene fusion system originally developed for E.coli for use in eukaryotic COS.M6 cells. We have fused PhoA and LacZ to the putative external and cytoplasmic loops of rat aquaporin 2 (AQP2), for which a model with six transmembrane domains was proposed previously. The fusion proteins were expressed in E.coli and COS.M6 cells and immunoblot analyses and enzyme activity assays were performed to localize the protein domains in both cell types. The data obtained in E.coli correlated mostly with the predictions of the six transmembrane domain model. However, two fusions were found to exhibit both high PhoA and high LacZ activity, thereby complicating the construction of a complete AQP2 model. In COS.M6 cells, the PhoA fusions were inactive. In contrast, the LacZ fusions succeeded and showed an activity pattern in complete agreement with the predictions of the six transmembrane domain model. Therefore, LacZ fusions can localize cytoplasmic loops in COS.M6 cells by means of a simple enzymatic assay with high reliability and may be used in future studies to develop topological models of other eukaryotic membrane proteins in their authentic cell systems.

Membrane targeting and determination of transmembrane topology of the human vasopressin V2 receptor.

The human vasopressin V2 receptor belongs to the large family of G-protein-coupled receptors, which possess seven transmembrane helices, an extracellular N terminus and an intracellular C terminus. We have determined the sequence requirements of the V2 receptor for membrane insertion and correct topology for the inner membrane of Escherichia coli with the PhoA/LacZ gene fusion system. In addition, we have studied the signals for its membrane insertion and correct topology for the membrane of the endoplasmic reticulum of the authentic eucaryotic transport system. To this end, we have extended the PhoA/LacZ gene fusion system for the first time to eucaryotic cells, i.e. transiently transfected COS.M6 cells. Truncated V2 receptor sequences were fused to PhoA and LacZ and expressed in both E. coli and COS.M6 cells. Cells were fractionated, and LacZ/PhoA activity assays and immunoblots were performed. We show here that a V2 receptor fragment consisting of the N terminus, the first transmembrane segment and the first cytoplasmic loop (71 amino acids) provided sufficient information for membrane insertion and correct orientation (extracellular N terminus) in both procaryotic and eucaryotic cells. Our data differ substantially from those obtained for the human beta2-adrenergic receptor expressed in E. coli (Lacatena, R. M., Cellini, A., Scavizzi, F., and Tocchini-Valentini, G. P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10521-10525). To establish correct topology, the beta2-adrenergic receptor requires a larger receptor portion, including the three N-terminal transmembrane segments and/or parts of the second cytoplasmic loop. The present data show that the observations made for the beta2-adrenergic receptor cannot be applied to G-protein-coupled receptors generally.

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.

Two novel mutations in the vasopressin V2 receptor gene in patients with congenital nephrogenic diabetes insipidus.

Families with congenital nephrogenic diabetes insipidus were analyzed with regard to mutations in the vasopressin V2 receptor gene. Family 1 shows an X-chromosomal recessive inheritance of the disease over 4 generations. A patient from this family was found to have a T-->A transversion at nucleotide 1095, predictive for a substitution of serine 167 (which is highly conserved among G-protein-coupled receptors), by threonine. Both the mutant and the normal allele were detected in the maternal genome. The patient's healthy brother was homozygous for the normal allele. The patient from family 2 showed a T-->C transition at nucleotide 727, predictive for a substitution of leucine 44 by proline. Analysis of the maternal genome revealed homozygosity for the normal allele. Thus a de novo mutation seems to have occurred. The nature and site of the mutation in family 2 suggest that it is responsible for the patient's disease.