Survival, neuron-like differentiation and functionality of mesenchymal stem cells in neurotoxic environment: the critical role of erythropoietin.

Mesenchymal stem cells (MSCs) can ameliorate symptoms in several neurodegenerative diseases. However, the toxic environment of a degenerating central nervous system (CNS) characterized by hypoxia, glutamate (Glu) excess and amyloid beta (Abeta) pathology may hamper the survival and regenerative/replacing capacities of engrafted stem cells. Indeed, human MSC (hMSC) exposed to hypoxia were disabled in (i) the capacity of their muscarinic receptors (mAChRs) to respond to acetylcholine (ACh) with a transient increase in intracellular [Ca(2+)], (ii) their capacity to metabolize Glu, reflected by a strong decrease in glutamine synthetase activity, and (iii) their survival on exposure to Glu. Cocultivation of MSC with PC12 cells expressing the amyloid precursor protein gene (APPsw-PC12) increased the release of IL-6 from MSC. HMSC exposed to erythropoietin (EPO) showed a cholinergic neuron-like phenotype reflected by increased cellular levels of choline acetyltransferase, ACh and mAChR. All their functional deficits observed under hypoxia, Glu exposure and APPsw-PC12 cocultivation were reversed by the application of EPO, which increased the expression of Wnt3a. EPO also enhanced the metabolism of Abeta in MSC by increasing their neprilysin content. Our data show that cholinergic neuron-like differentiation of MSC, their functionality and resistance to a neurotoxic environment is regulated and can be improved by EPO, highlighting its potential for optimizing cellular therapies of the CNS.

[Slim despite a genetic predisposition for obesity--influence of environmental factors as chance? A case report]. / Schlank trotz genetischer Prädisposition für Adipositas--Beeinflussung von Umweltfaktoren als Chance? Eine Kasuistik.

CASE HISTORY AND CLINICAL FINDINGS: A 4-year-old, slightly overweight (BMI 18 kg/m2, BMI SDS 1.29 approximately 90.1 (th) percentile) and otherwise healthy girl presented with accelerated linear growth (99.6 (th) percentile) and hyperphagia due to loss of satiety feeling. These findings are consistent with melanocortin-4 receptor ( MC4R) mutations. GENETIC ANALYSIS AND DIAGNOSIS: We found the partially inactivating mutation Arg (165)Trp by direct sequencing of the MC4R coding region. Interestingly, the patient's mother is also heterozygous for this mutation, but lean (BMI 19 kg/m2). TREATMENT AND COURSE: Carriers of MC4R mutations develop hyperphagia due to lack of satiety feeling as a result of central dysregulation. The reduction of energy intake and the encouragement of physical activity can be successful to control the body weight. CONCLUSION: Early diagnosis can promote lifestyle intervention to prevent the development of obesity even in the presence of a genetic predisposition.

Single-cell epithelial defects close rapidly by an actinomyosin purse string mechanism with functional tight junctions.

Restitution of single-cell defects, a frequent event in epithelia with high turnover, is poorly understood. Morphological and functional changes were recorded, using intravital time-lapse video microscopy, confocal fluorescence microscopy, and conductance scanning techniques. After artificial single-cell loss from an HT-29/B6 colonic cell monolayer, the basal ends of adjacent cells extended. Concurrently, the local conductive leak associated with the defect sealed with an exponential time course (from 0.48 +/- 0.05 microS 2 min post lesion to 0.17 +/- 0.02 microS 8 min post lesion, n = 17). Between 3 and 10 min post lesion, a band of actin arose around the gap, which colocalized with a ring of ZO-1 and occludin. Hence, tight junction proteins bound to the actin band facing the gap, and competent tight junctions assembled in the adjoining cell membranes. Closure and sealing were inhibited when actin polymerization was blocked by cytochalasin D, delayed following decrease of myosin-ATPase activity by butanedione monoxime, and blocked after myosin light chain kinase inhibition by ML-7. The Rho-associated protein kinase inhibitor Y-27632 did not affect restitution. After loosening of intercellular contacts in low Ca(2+) Ringer solution, the time course of restitution was not significantly altered. Albeit epithelial conductivity was 12-fold higher in low Ca(2+) Ringer solution than in controls, under both conditions the repaired epithelium assumed the same conductivity as distant intact epithelium. In conclusion, epithelial restitution of single-cell defects comprises rapid closure by an actinomyosin 'purse-string' mechanism and simultaneous formation of a functional barrier from tight junction proteins also associated with the purse string.

Nuclear envelope proteomics: novel integral membrane proteins of the inner nuclear membrane.

The nuclear envelope (NE) is one of the least characterized structures of eukaryotic cells. The study of its functional roles is hampered by the small number of proteins known to be specifically located to it. Here, we present a comprehensive characterization of the NE proteome. We applied different fractionation procedures and isolated protein subsets derived from distinct NE compartments. We identified 148 different proteins by 16-benzyl dimethyl hexadecyl ammonium chloride (16-BAC) gel electrophoresis and matrix-assisted laser desorption ionization (MALDI) mass spectrometry; among them were 19 previously unknown or noncharacterized. The identification of known proteins in particular NE fractions enabled us to assign novel proteins to NE substructures. Thus, our subcellular proteomics approach retains the screening character of classical proteomic studies, but also allows a number of predictions about subcellular localization and interactions of previously noncharacterized proteins. We demonstrate this result by showing that two novel transmembrane proteins, a 100-kDa protein with similarity to Caenorhabditis elegans Unc-84A and an unrelated 45-kDa protein we named LUMA, reside in the inner nuclear membrane and likely interact with the nuclear lamina. The utility of our approach is not restricted to the investigation of the NE. Our approach should be applicable to the analysis of other complex membrane structures of the cell as well.

The first activating TSH receptor mutation in transmembrane domain 1 identified in a family with nonautoimmune hyperthyroidism.

Sporadic and familial nonautoimmune hyperthyroidism are very rarely occurring diseases. Within the last years constitutively activating TSH receptor mutations were identified as one possible pathomechanism. Except for S281N in the extracellular N-terminal domain, all other germline mutations are located in the transmembrane domains 2, 3, 5, 6, and 7 of the TSH receptor, whereas no mutation was reported in transmembrane domains 1 and 4 to date. Here we report the first family with a constitutively active TSHR mutation in transmembrane domain 1 resulting in a substitution of the conserved Gly(431) for Ser. This mutation was found in the investigated patient, his father, and the paternal grandmother. As known from other familial cases of nonautoimmune hyperthyroidism, the age of onset of the disease was variable, ranging from early childhood in the patient and his father to adolescence in the grandmother. Functional characterization of this mutation showed a constitutive activation of the G(s)/adenylyl cyclase system. Moreover, this germline mutation also activates the G(q/11)/phospholipase C pathway. The importance of Gly(431) for receptor quiescence is supported further by introduction of other mutations at this position, all leading to constitutive receptor activity. Our data show now that constitutively activating mutations can be found in the entire transmembrane domain region of the TSH receptor, indicating the important role of all parts of the transmembrane domain region for maintaining the inactive receptor conformation.

Constitutively activating TSH-receptor mutations as a molecular cause of non-autoimmune hyperthyroidism in childhood.

BACKGROUND: The glycoprotein hormone TSH (thyroid-stimulating hormone) and its receptor, the TSH-receptor (TSHR), play a crucial role in thyroid growth and function. Constitutively activating germline mutations within the TSHR gene were identified in patients with sporadic or familial non-autoimmune hyperthyroidism. Inheritance of these mutations is autosomal dominant. PATIENTS AND METHODS: We investigated two patients with neonatal onset of non-autoimmune hyperthyroidism and two families in whom the child and one parent are affected. RESULTS: Hyperthyroidism was difficult to treat in all of these patients and was complicated by premature craniosynostosis. Sequencing of all exons of the TSHR gene in one family with hyperthyroidism revealed a mutation in exon 10 (T6321), which was first identified in toxic adenomas and found to constitutively activate the TSHR. In the other family, we identified a new mutation in the first membrane spanning segment (G431S). In both patients with sporadic hyperthyroidism, a heterozygous mutation in exon 9 (S281N) was detected. The functional characterization of S281N and G431S demonstrated that both mutants were constitutively active. Therefore, these mutations are the molecular cause of non-autoimmune hyperthyroidism in the patients. CONCLUSIONS: For patients suffering from non-autoimmune hyperthyroidism, screening for mutations and their functional characterization is recommended. In case of an ineffective hyperthyroidism treatment, thyroidectomy should be performed to prevent lengthy anti-thyroid drug treatment and complications like premature craniosynostosis.

Gonadotropin-releasing hormone receptor initiates multiple signaling pathways by exclusively coupling to G(q/11) proteins.

The agonist-bound gonadotropin-releasing hormone (GnRH) receptor engages several distinct signaling cascades, and it has recently been proposed that coupling of a single type of receptor to multiple G proteins (G(q), G(s), and G(i)) is responsible for this behavior. GnRH-dependent signaling was studied in gonadotropic alphaT3-1 cells endogenously expressing the murine receptor and in CHO-K1 (CHO#3) and COS-7 cells transfected with the human GnRH receptor cDNA. In all cell systems studied, GnRH-induced phospholipase C activation and Ca(2+) mobilization was pertussis toxin-insensitive, as was GnRH-mediated extracellular signal-regulated kinase activation. Whereas the G(i)-coupled m2 muscarinic receptor interacted with a chimeric G(s) protein (G(s)i5) containing the C-terminal five amino acids of Galpha(i2), the human GnRH receptor was unable to activate the G protein chimera. GnRH challenge of alphaT3-1, CHO#3 and of GnRH receptor-expressing COS-7 cells did not result in agonist-dependent cAMP formation. GnRH challenge of CHO#3 cells expressing a cAMP-responsive element-driven firefly luciferase did not result in increased reporter gene expression. However, coexpression of the human GnRH receptor and adenylyl cyclase I in COS-7 cells led to clearly discernible GnRH-dependent cAMP formation subsequent to GnRH-elicited rises in [Ca(2+)](i). In alphaT3-1 and CHO#3 cell membranes, addition of [alpha-(32)P]GTP azidoanilide resulted in GnRH receptor-dependent labeling of Galpha(q/11) but not of Galpha(i), Galpha(s) or Galpha(12/13) proteins. Thus, the murine and human GnRH receptors exclusively couple to G proteins of the G(q/11) family. Multiple GnRH-dependent signaling pathways are therefore initiated downstream of the receptor/G protein interface and are not indicative of a multiple G protein coupling potential of the GnRH receptor.

Compound deletion of the rhoGAP C1 and V2 vasopressin receptor genes in a patient with nephrogenic diabetes insipidus.

The function of small GTPases is fine-tuned by a complex network of regulatory proteins such as GTPase-activating proteins. The C1 gene at Xq28 encodes a protein assumed to function as a Rho GTPase-activating protein (rhoGAP). Characterization of the molecular defect causing X-linked nephrogenic diabetes insipidus (NDI) in a patient revealed a submicroscopic deletion of a 21.5-kb genomic fragment encompassing the entire arginine-vasopressin V2 receptor gene (AVPR2) and most of the C1 gene locus. In the absence of detailed information about the physiological relevance and specific functions of rhoGAP C1, a thorough clinical and laboratory investigation of the patient was performed. Besides clearly defined NDI symptoms caused by deletion of the AVPR2 gene, no major morphological abnormalities as determined by physical examination, radiography, ultrasound, and computed tomographic scan were detected. Extensive analysis of blood chemical, enzyme, and hormone values over a period of 16 years showed no deviations from normal ranges. On the basis of our observations, the rhoGAP C1 protein is not essential for normal development in the human. Because of a predominant expression pattern of the C1 gene in hematopoietic cells, we focused on immunologic and hematologic laboratory parameters of the affected boy and the mother who was found to be heterozygous. Differential white cell counts, including lymphocyte typing, determination of lymphokines, cytokines, and immunoglobulins, as well as numerous leukocyte function tests, showed no pathological findings. Therefore, we postulate that the loss of rhoGAP C1 function is most likely compensated by other members of the GAP family.

Conserved extracellular cysteine pair in the M3 muscarinic acetylcholine receptor is essential for proper receptor cell surface localization but not for G protein coupling.

Most G protein-coupled receptors contain a conserved pair of extracellular cysteine residues that are predicted to form a disulfide bond linking the first and second extracellular loops. Previous studies have shown that this disulfide bond may be critical for ligand binding, receptor activation, and/or proper receptor folding. However, the potential importance of the two conserved cysteine residues for proper receptor cell surface localization has not been investigated systematically. To address this issue, we used the rat M3 muscarinic receptor as a model system. Most studies were carried out with a modified version of this receptor subtype (lacking potential N-glycosylation sites and the central portion of the third intracellular loop) that could be readily detected via western blot analysis. Cys-->Ala mutant receptors were generated, transiently expressed in COS-7 cells, and then examined for their subcellular distribution and functional properties. ELISA and immunofluorescence studies showed that the presence of both conserved cysteine residues (corresponding to C140 and C220 in the rat M3 muscarinic receptor sequence) is required for efficient expression of the M3 muscarinic receptor on the cell surface. On the other hand, these residues were found not to be essential for protein stability (determined via immunoblotting) and receptor-mediated G protein activation (studied in second messenger assays). These results shed new light on the functional role of the two extracellular cysteine residues present in most G protein-coupled receptors.

Alterations of neonatal thyroid function.

Recent progress has been made in understanding the pathogenesis of neonatal thyroid disorders. Autosomal recessive inheritance of mutations of the thyroid peroxidase and thyroglobulin genes has been described in some patients with congenital hypothyroidism (CH) and a family history of CH. Autosomal recessive inheritance of mutations of the thyrotrophin (TSH) receptor gene has also been reported in patients with CH and thyroid hypoplasia, and autosomal dominant mutations of the PAX8 gene have been described in patients with different forms of thyroid dysgenesis. These discoveries are important for patients with CH diagnosed by neonatal screening, as these patients will have normal fertility. The molecular genetic analysis of mutations of the TSH gene in patients with familial and sporadic cases of isolated central CH, who are missed by TSH screening programmes, now enables rapid diagnosis and appropriate therapy in the neonate. In newborn infants with severe non-autoimmune hyperthyroidism, autosomal dominant gain-of-function mutations in the TSH receptor gene have been demonstrated. In these patients, molecular genetic studies are extremely helpful in therapeutic decision making, as early thyroid ablation is the only effective treatment that avoids the sequelae of long-term hyperthyroidism. Molecular genetic studies are therefore useful in the diagnostic work-up of neonatal thyroid alterations.

Role of the third intracellular loop for the activation of gonadotropin receptors.

Hyperfunctional endocrine thyroid and testicular disorders can frequently be traced back to gainof-function mutations in glycoprotein hormone receptor genes. Deletion mutations in the third intracellular (i3) loop of the TSH receptor have recently been identified as a cause of constitutive receptor activity. To examine whether the underlying mechanism of receptor activation applies to all glycoprotein hormone receptors, we created deletion mutations in the LH and FSH receptors. In analogy to the situation with the TSH receptor, a deletion of nine amino acids resulted in constitutive activity irrespective of the location of deletions within the i3 loop of the LH receptor. In contrast, only one (delta563-566) of four different 4-amino acid deletion mutants displayed agonist-independent activity. Systematic examination of the structural requirements for this effect in the delta563-566 mutant revealed that only deletions including D564 resulted in constitutive receptor activity. Replacement of D564 by G, K, and N led to agonist-independent cAMP formation while introduction of a negatively charged E silenced constitutive receptor activity, indicating that an anionic amino acid at this position may be required to maintain an inactive receptor conformation. Insertion of A residues up- and downstream of D564 did not perturb receptor quiescence, showing that a certain degree of spatial freedom of the negatively charged amino acid within the context of the i3 loop is well tolerated. In contrast to the results obtained with the LH receptor, deletion of the corresponding D567 from the i3 loop of the FSH receptor did not cause constitutive receptor activation, highlighting significant differences in the activation mechanism of gonadotropin receptors.

Adenoviral gene transfer of the human V2 vasopressin receptor improves contractile force of rat cardiomyocytes.

BACKGROUND: In congestive heart failure, high systemic levels of the hormone arginine vasopressin (AVP) result in vasoconstriction and reduced cardiac contractility. These effects are mediated by the V1 vasopressin receptor (V1R) coupled to phospholipase C beta-isoforms. The V2 vasopressin receptor (V2R), which promotes activation of the Gs/adenylyl cyclase system, is physiologically expressed in the kidney but not in the myocardium. Expression of a recombinant V2R (rV2R) in the myocardium could result in a positive inotropic effect via the endogenous high concentrations of AVP in heart failure. METHODS AND RESULTS: A recombinant adenovirus encoding the human V2R (Ad-V2R) was tested for its ability to modulate the cardiac Gs/adenylyl cyclase system and to potentiate contractile force in rat ventricular cardiomyocytes and in H9c2 cardiomyoblasts. Ad-V2R infection resulted in a virus concentration-dependent expression of the transgene and led to a marked increase in cAMP formation in rV2R-expressing cardiomyocytes after exposure to AVP. Single-cell shortening measurements showed a significant agonist-induced contraction amplitude enhancement, which was blocked by the V2R antagonist, SR 121463A. Pretreatment of Ad-V2R-infected cardiomyocytes with AVP led to desensitization of the rV2R after short-term agonist exposure but did not lead to further loss of receptor function or density after long-term agonist incubation, thus demonstrating resistance of the rV2R to downregulation. CONCLUSIONS: Adenoviral gene transfer of the V2R in cardiomyocytes can modulate the endogenous adenylyl cyclase-signal transduction cascade and can potentiate contraction amplitude in cardiomyocytes. Heterologous expression of cAMP-forming receptors in the myocardium could lead to novel strategies in congestive heart failure by bypassing the desensitized beta-adrenergic receptor signaling.

A conserved tyrosine residue (Y601) in transmembrane domain 5 of the human thyrotropin receptor serves as a molecular switch to determine G-protein coupling.

In the human thyroid, the wild-type thyrotropin receptor (TSHR) couples to adenylyl cyclase and phospholipase C and constitutively increases intracellular cAMP levels. The first human TSHR sequence submitted differs from subsequently cloned wild-type receptors by an exchange of a conserved Y residue within transmembrane domain 5 (TM5) for an H residue. We did not detect the Y601H mutant in 263 European individuals, but confirmed the homozygous occurrence of TSHR-Y601. Expression of TSHR-Y601H in COS-7 cells revealed a loss of constitutive cAMP production and selective lack of TSH-induced phosphoinositide hydrolysis, whereas agonist-induced cAMP formation remained unaltered. Analysis of several mutant receptors (Y601A, Y601D, Y601F, Y601K, Y601P, Y601S, Y601W, Y601Delta) did not show restoration of constitutive activity and dual signaling, thus suggesting a functional role of a properly spaced hydroxyl group at position 601. Molecular modeling revealed that the formation of a hydrogen bond between the hydroxyl group of Y601 in TM5 and the carbonyl oxygen of A623 in the peptide backbone of TM6 is critical for the receptor to adopt active conformations that impart wild-type signaling properties. Our findings indicate that multiple active receptor states underlie coupling of a G-protein-coupled receptor to different G-proteins.

Enhancement of phospholipase D activity following baculovirus and adenovirus infection in Sf9 and COS-7 cells.

In order to purify the human phospholipase D1 (hPLD1) for analysis of its functional properties, we applied a baculovirus-based high-expression system. As expected, Sf9 cells infected with a baculovirus encoding for the hPLD1 displayed a 7.5-fold increase in PLD activity compared to uninfected cells. Sf9 cells infected with the wild-type (WT) and other recombinant baculoviruses were used as an expression control. Surprisingly, all baculoviruses tested led to a 3-5 fold increase in basal PLD activity when compared to uninfected cells. To further characterize the nature of the increased PLD activity, the influence of ADP-ribosylation factor (ARF) and phorbol 12-myristate 13-acetate (PMA) was studied. In contrast to membranes containing the hPLD1, the PLD activity in membranes from uninfected and WT-infected Sf9 cells was not stimulated by ARF. PMA did not affect the increase in PLD activity in any case. To further study whether the virus-mediated increase in PLD activity is a more general phenomenon, we infected COS-7 cells with recombinant and WT adenoviruses. Only the infection with the WT adenovirus resulted in an approx. 2-fold increase in PLD activity. Our results demonstrate for the first time that a viral infection elevates the PLD activity in insect and mammalian cells.

Deletions in the third intracellular loop of the thyrotropin receptor. A new mechanism for constitutive activation.

Gain-of-function mutations of the thyrotropin receptor (TSHR) gene have been invoked as one of the major causes of toxic thyroid adenomas. In a toxic thyroid nodule, we recently identified a 9-amino acid deletion (amino acid positions 613-621) within the third intracellular (i3) loop of the TSHR resulting in constitutive receptor activity. This finding exemplifies a new mechanism of TSHR activation and raises new questions concerning the function of the i3 loop. Because the i3 loop is thought to be critical for receptor/G protein interaction in many receptors, we systematically reexamined the role of the TSHR's i3 loop for G protein coupling. Thus, various deletion mutants were generated and functionally characterized. We identified an optimal deletion length responsible for constitutive activity. If the number of deleted amino acids was reduced, elevated basal cAMP accumulation was found to be concomitantly diminished. Expansion of the deletion dramatically impaired cell surface expression of the receptor. Shifting the deletion toward the N terminus of the i3 loop resulted in unaltered strong constitutive receptor activity. In contrast, translocation of the deletion toward the C terminus led to significantly reduced basal cAMP formation, most probably due to destruction of a conserved cluster of amino acids. In this study, we show for the first time that amino acid deletions within the i3 loop of a G protein-coupled receptor result in constitutive receptor activity. In the TSHR, 75% of the i3 loop generally assumed to play an essential role in G protein coupling can be deleted without rendering the mutant receptor unresponsive to thyrotropin. These findings support a novel model explaining the molecular events accompanying receptor activation by agonist.

Molecular aspects of vasopressin receptor function.

The molecular mechanisms governing the G protein coupling selectivity of different members of the vasopressin receptor family were studied by using a combined molecular genetic/biochemical approach. While the V1a and V1b vasopressin receptors are selectively linked to G proteins of the Gq/11 class, the V2 vasopressin receptor is preferentially coupled to Gs. Systematic functional analysis of V1a/V2 hybrid receptors showed that the second intracellular loop of the V1a receptor is required and sufficient for efficient coupling to Gq/11, whereas the third intracellular loop of the V2 receptor is required and sufficient for coupling to Gs. By using a strategy involving the coexpression of the wild type V1a receptor with chimeric G protein alpha s/alpha q subunits, two C-terminal alpha q/11 residues were identified that are critical for proper receptor recognition. We previously demonstrated -in transiently transfected COS-7 cells- that selected mutant V2 vasopressin receptors (all of which have been identified in X-linked nephrogenic diabetes insipidus patients) containing inactivating mutations in the C-terminal third of the receptor protein (including missense, frameshift, or nonsense mutations) can be functionally rescued by coexpression with a C-terminal V2 receptor fragment (V2-tail) spanning the region where the various mutations occur. Co-immunoprecipitation experiments and a newly developed sandwich ELISA revealed that the V2-tail polypeptide directly interacts with the mutant V2 receptors thus creating a functional receptor protein. To study the potential therapeutic usefulness of these findings, CHO cell lines stably expressing low levels of functionally inactive mutant V2 vasopressin receptors (E242stop, Y280C, and W284stop) were created and infected with a recombinant adenovirus coding for the V2-tail polypeptide. Following adenovirus infection, arginine vasopressin (AVP) gained the ability to stimulate cAMP formation in all CHO cell clones studied. Adenovirus-mediated gene transfer also proved to be a highly efficient method to achieve expression of the V2-tail fragment (as well as of the wild type V2 vasopressin receptor) in MDCK renal tubular cells. We therefore speculate that the targeted expression of receptor fragments in vivo may represent a novel strategy in the treatment of human diseases caused by inactivating mutations in distinct G protein-coupled receptors.

Reconstitution of mutant V2 vasopressin receptors by adenovirus-mediated gene transfer. Molecular basis and clinical implication.

Recent studies with transfected COS-7 cells have shown that functionally inactive mutant V2 vasopressin receptors (occurring in patients with nephrogenic diabetes insipidus) can be functionally rescued by coexpression of a carboxy-terminal V2 receptor fragment (V2-tail) spanning the region where various mutations occur [Schöneberg, T., J. Yun, D. Wenkert, and J. Wess. 1996. EMBO (Eur. Mol. Biol. Organ.) J. 15:1283-1291]. In this study, we set out to characterize the underlying molecular mechanism. Using a coimmunoprecipitation strategy and a newly developed sandwich ELISA system, a direct and highly specific interaction between the mutant V2 vasopressin receptor proteins and the V2-tail polypeptide was demonstrated. To study the potential therapeutic usefulness of these findings, Chinese hamster ovary (CHO) cell lines stably expressing low levels of functionally inactive mutant V2 vasopressin receptors were created and infected with a recombinant adenovirus carrying the V2-tail gene fragment. After adenovirus infection, vasopressin gained the ability to stimulate cAMP formation with high potency and efficacy in all CHO cell clones studied. Moreover, adenovirus-mediated gene transfer also proved to be a highly efficient method for achieving expression of the V2-tail fragment (as well as the wild-type V2 receptor) in Madin-Darby canine kidney tubular cells. Taken together, these studies clarify the molecular mechanisms by which receptor fragments can restore function of mutationally inactivated G protein-coupled receptors and suggest that adenovirus-mediated expression of receptor fragments may lead to novel strategies for the treatment of a variety of human diseases.

Inhibition of gonadotropin-releasing hormone receptor signaling by expression of a splice variant of the human receptor.

GnRH binds to a specific G protein-coupled receptor in the pituitary to regulate synthesis and secretion of gonadotropins. Using RT-PCR and human pituitary poly(A)+ RNA as a template, the full-length GnRH receptor (wild type) and a second truncated cDNA characterized by a 128-bp deletion between nucleotide positions 522 and 651 were cloned. The deletion causes a frame shift in the open reading frame, thus generating new coding sequence for further 75 amino acids. The truncated cDNA arises from alternative splicing by accepting a cryptic splicing acceptor site in exon 2. Distinct translation products of approximately 45-50 and 42 kDa were immunoprecipitated from COS-7 cells transfected with cDNA coding for wild type GnRH receptor and the truncated splice variant, respectively. Immunocytochemical and enzyme-linked immunosorbent assay studies revealed a membranous expression pattern for both receptor isoforms. Expression of the splice variant, however, occurred at a significantly lower cell surface receptor density. In terms of ligand binding and phospholipase C activation, the wild type receptor showed characteristics of a typical GnRH receptor, whereas the splice variant was incapable of ligand binding and signal transduction. Coexpression of wild type and truncated proteins in transiently or stably transfected cells, however, resulted in impaired signaling via the wild type receptor by reducing maximal agonist-induced inositol phosphate accumulation. The inhibitory effect depended on the amount of splice variant cDNA cotransfected and was specific for the GnRH receptor because signaling via other G(q/11)-coupled receptors, such as the thromboxane A2, M5 muscarinic, and V1 vasopressin receptors, was not affected. Immunological studies revealed that coexpression of the wild type receptor and the truncated splice variant resulted in impaired insertion of the wild type receptor into the plasma membrane. Thus, expression of truncated receptor proteins may highlight a novel principle of specific functional inhibition of G protein-coupled receptors.

Functional characterization of five V2 vasopressin receptor gene mutations.

COS7 cells were transiently transfected with plasmids encoding mutant forms of the V2 vasopressin receptors corresponding to mutations [Y280C, L292P, R337stop, V277A, and G12E (the latter found in the same kindred with L292P)] recently identified in subjects with X-linked nephrogenic diabetes insipidus (NDI). cAMP response to dDAVP and AVP, saturation binding experiments with [3H]-AVP, immunofluorescence, and indirect ELISA studies were performed to characterize the functional consequences of these mutations. The Y280C, L292P, and R337stop mutant V2 receptors show substantially decreased cell surface expression and are functionally inactive. The V277A mutant receptor, though well expressed at the cell surface as seen by immunofluorescence and ELISA and having a dissociation constant with AVP similar to the wild type receptor, was functionally less active as seen by a substantially decreased receptor number (Bmax) and reduced cAMP stimulation by dDAVP. The G12E mutant was functionally the same as the wild type V2 receptor in both cAMP stimulation and binding. These results provide insight into residues critical for V2 receptor expression and function and also provide direct evidence that Y280C, L292P, R337stop and V277A mutations are the cause of X-linked NDI in affected subjects.

Functional rescue of mutant V2 vasopressin receptors causing nephrogenic diabetes insipidus by a co-expressed receptor polypeptide.

Inactivating mutations in distinct G protein-coupled receptors (GPCRs) are currently being identified as the cause of a steadily growing number of human diseases. Based on previous studies showing that GPCRs are assembled from multiple independently stable folding units, we speculated that such mutant receptors might be functionally rescued by 'supplying' individual folding domains that are lacking or misfolded in the mutant receptors, by using a co-expression strategy. To test the feasibility of this approach, a series of nine mutant V2 vasopressin receptors known to be responsible for X-linked nephrogenic diabetes insipidus were used as model systems. These mutant receptors contained nonsense, frameshift, deletion or missense mutations in the third intracellular loop or the last two transmembrane helices. Studies with transfected COS-7 cells showed that none of these mutant receptors, in contrast to the wild-type V2 receptor, was able to bind detectable amounts of the radioligand, [3H]arginine vasopressin, or to activate the G(S)/adenylyl cyclase system. Moreover, immunological studies demonstrated that the mutant receptors were not trafficked properly to the cell surface. However, several of the nine mutant receptors regained considerable functional activity upon co-expression with a C-terminal V2 receptor peptide spanning the sequence where the various mutations occur. In many cases, the restoration of receptor activity by the co-expressed receptor peptide was accompanied by a significant increase in cell surface receptor density. These findings may lead to the design of novel strategies in the treatment of diseases caused by inactivating mutations in distinct GPCRs.