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Biomedical subjects

G Schultz

Publications and source records attributed to G Schultz.

At least 73 records · Page 4Linked to original sources
PubMed1997

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.

Adenoviridae↗
PubMed1997

A betagamma dimer derived from G13 transduces the angiotensin AT1 receptor signal to stimulation of Ca2+ channels in rat portal vein myocytes.

A G protein composed of alpha13, beta1, and gamma3 subunits selectively couples the angiotensin AT1A receptors to increase cytoplasmic Ca2+ concentration ([Ca2+]i) in rat portal vein myocytes (Macrez-Leprêtre, N., Kalkbrenner, F., Morel, J. L., Schultz, G., and Mironneau, J. (1997) J. Biol. Chem. 272, 10095-10102). We show here that Gbetagamma transduces the signal leading to stimulation of L-type Ca2+ channels. Intracellular dialysis through the patch pipette of a carboxyl-terminal anti-betacom antibody and a peptide corresponding to the Gbetagamma binding region of the beta-adrenergic receptor kinase 1 inhibited the stimulation of Ca2+ channels and the increase in [Ca2+]i evoked by angiotensin II. The Gbetagamma binding peptide did not prevent the dissociation of the heterotrimeric G protein into its subunits, as it did not block activation of phospholipase C-beta by Galphaq in response to stimulation of alpha1-adrenoreceptors. Transient overexpression of the beta-adrenergic receptor kinase 1 fragment and of Galpha subunits also inhibited the angiotensin II-induced increase in [Ca2+]i. Both anti-alpha13 antibody and carboxyl-terminal alpha13 peptide abrogated the angiotensin II-induced stimulation of Ca2+ channels. We conclude that activation of angiotensin AT1 receptors requires all three alpha, beta, and gamma subunits of G13 for receptor-G protein interaction, whereas the transduction of the signal to L-type Ca2+ channels is mediated by Gbetagamma.

Amino Acid Sequence↗
PubMed1997

G protein heterotrimer Galpha13beta1gamma3 couples the angiotensin AT1A receptor to increases in cytoplasmic Ca2+ in rat portal vein myocytes.

The subunit composition of angiotensin AT1 receptor-activated G protein was identified by using antisense oligonucleotide injection into the nucleus of rat portal vein myocytes. In these cells, we have previously shown that increases in the cytoplasmic calcium concentration ([Ca2+]i) induced by activation of angiotensin AT1 receptors were dependent on extracellular Ca2+ entry by L-type Ca2+ channels and subsequent Ca2+-induced Ca2+ release from the intracellular stores. The angiotensin AT1 receptor-activated increases in [Ca2+]i were selectively inhibited by injection of antisense oligonucleotides directed against the mRNAs coding for the alpha13, beta1, and gamma3 subunits. A correlating reduction in Galpha13, Gbeta1, and Ggamma3 protein expression was confirmed by immunocytochemistry. In addition, anti-alpha13 antibody and synthetic peptide corresponding to the carboxyl terminus of the Galpha13 subunit inhibited, in a concentration-dependent manner, the angiotensin AT1 receptor-mediated Ca2+ response. Reverse transcription-polymerase chain reaction analysis showed that only the angiotensin AT1A receptor was expressed in rat portal vein smooth muscle. Furthermore, injection of anti-AT1A oligonucleotides selectively inhibited the angiotensin II-induced increase in [Ca2+]i. We conclude that the receptor-activated signal leading to increases in [Ca2+]i is transduced by the heterotrimeric G13 protein composed of alpha13/beta1/gamma3 subunits and that the carboxyl terminus of the Galpha13 subunit interacts with the angiotensin AT1A receptor.

Angiotensin II↗
PubMed1997

Multiple G-protein coupling of the dog thyrotropin receptor.

We investigated, in dog thyroid membranes, the ability of the dog thyrotropin (TSH) receptor to interact with the endogenous G proteins expressed in this tissue. Activation of the receptor led to increased incorporation of the photoreactive GTP analog [alpha-(32)P]GTP azidoanilide into immunoprecipitated alpha subunits of three G protein families: G(s), G(q/11), G(i/o). This effect was not due to a general loss of receptor G protein specificity since carbamylcholine, in the same membrane preparations, only stimulated the binding of the GTP analog to the alpha subunits of G(q/11) proteins. To investigate the multiple coupling of the dog TSH receptor in intact cells, cyclic AMP accumulation, IP(3) formation and (45)Ca2+ efflux experiments were performed. When thyrocytes were pretreated with pertussis toxin (PTX), the TSH receptor-mediated accumulation of cAMP increased by approximately 45% with TSH at 1 mU/ml, suggesting that the TSH receptor coupled to both G(s) and G(i) in vivo. On the other hand, no increase in IP(3) accumulation nor Ca2+ efflux was observed in the presence of thyrotropin. These data in intact cells are thus in contradiction with those obtained in membranes, suggesting that receptor-mediated transmembrane signalling may implicate a specificity which itself may reflect a localization and organization of the different components (receptors, G proteins, ...) in the plasma membrane of intact cells. As in some cells, G(i) activates mitogenesis by hormone activated G-protein-coupled receptors, we tested its role in the stimulation by TSH of the proliferation of thyrocytes. This was not affected by PTX, suggesting that the mitogenic effect of TSH does not involve G(i)-proteins.

Animals↗
PubMed1997

Distinct functions of Gq and G11 proteins in coupling alpha1-adrenoreceptors to Ca2+ release and Ca2+ entry in rat portal vein myocytes.

In this study, we identified the subunit composition of Gq and G11 proteins coupling alpha1-adrenoreceptors to increase in cytoplasmic Ca2+ concentration ([Ca2+]i) in rat portal vein myocytes maintained in short-term primary culture. We used intranuclear antisense oligonucleotide injection to inhibit selectively the expression of subunits of G protein. Increases in [Ca2+]i were measured in response to activation of alpha1-adrenoreceptors, angiotensin AT1 receptors, and caffeine. Antisense oligonucleotides directed against the mRNAs coding for alphaq, alpha11, beta1, beta3, gamma2, and gamma3 subunits selectively inhibited the increase in [Ca2+]i activated by alpha1-adrenoreceptors. A corresponding reduction of the expression of these G protein subunits was immunochemically confirmed. In experiments performed in Ca2+-free solution only cells injected with anti-alphaq antisense oligonucleotides displayed a reduction of the alpha1-adrenoreceptor-induced Ca2+ release. In contrast, in Ca2+-containing solution, injection of anti-alpha11 antisense oligonucleotides suppressed the alpha1-adrenoreceptor-induced stimulation of the store-operated Ca2+ influx. Agents that specifically bound Gbetagamma subunits (anti-betacom antibody and overexpression of a beta-adrenergic receptor kinase carboxyl-terminal fragment) had no effect on the alpha1-adrenoreceptor-induced signal transduction. Taken together, these results suggest that alpha1-adrenoreceptors utilize two different Galpha subunits to increase [Ca2+]i. Galphaq may activate phosphatidylinositol 4,5-bisphosphate hydrolysis and induce release of Ca2+ from intracellular stores. Galpha11 may enhance the Ca2+-activated Ca2+ influx that replenishes intracellular Ca2+ stores.

Animals↗
PubMed1997

Functions of conserved cysteines of soluble guanylyl cyclase.

Soluble guanylyl cyclase (sGC), a heme-containing heterodimeric enzyme, is stimulated by NO and catalyzes the formation of the intracellular signaling molecule cGMP. Cysteine residues of sGC have been considered to be important as they were thought to play a significant role in the regulation of the enzyme. The aim of this study was to investigate the possible function of conserved cysteine residues of sGC. Fifteen conserved cysteine residues on sGC were point-mutated to serine, using site-directed mutagenesis. All of the resulting recombinant enzymes were able to synthesize cGMP. Mutation of two cysteines located in the N-terminal, putative heme-binding region of the beta1 subunit yielded proteins that were insensitive to NO. Spectrophotometric analysis of the NO-insensitive mutants purified from Sf9 cells revealed a loss of the prosthetic heme group. Both mutants could be reconstituted with heme and, as a consequence, NO sensitivity of the mutants was restored. Our data show that mutation of two cysteines of the beta1 subunit (Cys-78 and Cys-214) reduces the affinity of sGC for heme. Mutation of the corresponding cysteines on the alpha1 subunit did not alter NO responsiveness, indicating that heme-binding is mainly a feature of the N-terminal domain of the beta1 subunit.

Animals↗
PubMed1997

Expression of epidermal growth factor receptor in gestational trophoblastic diseases.

Gestational trophoblastic disease is an abnormal condition of the placenta, the incidence of which is very high in the State of Kerala, India. The biology of this disease is vague and methods to determine the malignant potential are limited. Placentae of normal (50) and molar pregnancies (95), including 32 showing persistent disease, were used for the study. Epidermal growth factor (EGFR), expression was evaluated by immunohistochemistry using monoclonal antibody, and quantified using the 125I-EGF-binding assay. EGFR was expressed more strongly in molar placentae than in normal placentae of similar gestational age, and was strong in the molar placentae of all gestational ages, whereas expression was mainly restricted to the first and second trimesters in normal placentae. Moles with early presenting symptoms (before 16 weeks) were at a higher risk of developing persistent disease. To conclude, the immunohistochemical study shows high expression of EGFR in early normal placentae and in moles, linking its role to the proliferative and differentiating activity of trophoblasts. Tumours with a histological diagnosis of invasive moles and choriocarcinoma showed very strong binding of EGFR. The present observations also suggest the possibility of mutated EGF receptors being present in persistent trophoblastic disease.

Adult↗
PubMed1997

Phantom limbs in people with congenital limb deficiency or amputation in early childhood.

It is widely believed that people who are congenitally limb-deficient or suffer a limb amputation at an early age do not experience phantom limbs. The present study reports on a sample of 125 people with missing limbs and documents phantom experiences in 41 individuals who were either born limb-deficient (n = 15) or underwent amputation before the age of 6 years (n = 26). These cases provide evidence that phantom limbs are experienced by at least 20% of congenitally limb-deficient subjects and by 50% of subjects who underwent amputations before the age of 6 years. The phantoms are detailed and can be described in terms of size, shape, position, movement and temporal properties. The perceptual qualities of the phantoms can also be described by sensory descriptors and are reported as painful by 20% of subjects with phantoms in the congenital limb deficient group and 42% of young amputees. It is argued that these phantom experiences provide evidence of a distributed neural representation of the body that is in part genetically determined.

Adolescent↗
PubMed1997

Functional and structural complexity of signal transduction via G-protein-coupled receptors.

A prerequisite for the maintenance of homeostasis in a living organism is fine-tuned communication between different cells. The majority of extracellular signaling molecules, such as hormones and neurotransmitters, interact with a three-protein transmembrane signaling system consisting of a receptor, a G protein, and an effector. These single components interact sequentially and reversibly. Considering that hundreds of G-protein-coupled receptors interact with a limited repertoire of G proteins, the question of coupling specificity is worth considering. G-protein-mediated signal transduction is a complex signaling network with diverging and converging transduction steps at each coupling interface. The recent realization that classical signaling pathways are intimately intertwined with growth-factor-signaling cascades adds another level of complexity. Elaborate studies have significantly enhanced our knowledge of the functional anatomy of G-protein-coupled receptors, and the concept has emerged that receptor function can be modulated with high specificity by coexpressed receptor fragments. These results may have significant clinical impact in the future.

Animals↗
PubMed1997

Mutations of the human thyrotropin receptor gene causing thyroid hypoplasia and persistent congenital hypothyroidism.

The pathogenesis of congenital hypothyroidism due to thyroid dysgenesis is still unknown. A point mutation in the TSH receptor (TSHR) of the hypothyroid hyt/hyt mouse invoked the TSHR as a candidate gene for congenital hypothyroidism. Therefore, we screened for mutations in the TSHR gene in patients with congenital hypothyroidism and hypoplasia of the gland. In one girl detected in neonatal screening with the confirmed diagnosis of permanent congenital hypothyroidism with reduced thyroid volume, two novel mutations in the TSHR gene were identified. Single strand conformational polymorphism and subsequent DNA sequencing studies of a fragment of the TSHR gene showed that the patient is a compound heterozygote for 2 loss of function mutations in exon 10 of the TSHR gene. In the mutant maternal allele, 18 nucleotides (positions 1217-1234) are deleted, and 4 novel bp are inserted, resulting in a frame-shift and premature termination of the coding sequence. Transfection studies showed that this truncated TSHR was trapped intracellularly and completely lacked cell surface expression. The paternal gene harbors a missense mutation at nucleotide position 1170, leading to the exchange of the highly conserved C-390 for a W residue. This alteration resulted in a drastic loss of affinity and potency of TSH acting at the mutant compared to the wild-type receptor. In contrast to the published loss of function mutations of the TSHR leading to euthyroid hyperthyrotropinemia, the two new mutations lead to persistent congenital hypothyroidism and defective organ development. Further studies will have to analyze to what extent TSHR mutations are involved in the pathogenesis of congenital hypothyroidism as opposed to other genetic or environmental factors.

Amino Acid Sequence↗
PubMed1997

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.

Alternative Splicing↗
PubMed1997

Treatment of bleb leaks with transforming growth factor-beta in the rabbit model.

PURPOSE: The mechanism through which peribleb injection of autologous blood results in resolution of bleb leak in the rabbit model remains unclear. This study evaluates the clinical and histologic effects of peribleb injection of transforming growth factor-beta (TGF-beta) after leak induction in mitomycin-C-treated blebs. METHOD: Posterior lip sclerectomies treated with mitomycin-C were created in New Zealand White rabbits. On postoperative day 7, a standardized stab incision was performed on all blebs, and the eyes were randomized to receive a peribleb injection of either TGF-beta or of a balanced salt solution. RESULTS: Injection of TGF-beta was associated with the resolution of bleb leak and maintenance of a functioning bleb in 50% (4 of 8) of treated eyes. The remaining TGF-beta-treated eyes and control eyes demonstrated continued bleb leaks or bleb failures with intraocular pressure returning to preoperative levels. Histologic examination revealed increased peribleb cellularity and denser collagen deposition in the TGF-beta-treated eyes compared with that observed in control eyes. CONCLUSIONS: Peribleb TGF-beta injections may contribute to healing bleb leaks, but the injections do not appear in this model to be as useful as whole-blood injections.

Animals↗
PubMed1997

Expression of epidermal growth factor in gestational trophoblastic disease (GTD).

Gestational Trophoblastic Disease (GTD) is an abnormal condition of the placenta, the incidence of which is very high in the State of Kerala, India. The biology of the disease is vague and methods to determine the malignant potentialis limited. Placentae of normal (50) and molar pregnancy (95) including 32 patients showing persistent disease were used in this study. EGF expression was analyzed by immunohistochemistry using monoclonal antibody to EGF and quantitated using isotope labelled antibody to EGF. EGF was expressed more strongly in molar placentae in all gestational ages in comparison with normal placentae of similar gestational age, the expression being restricted mainly to first and second trimesters in normal placentae. Moles with early presenting symptoms (< 12 weeks) were at a higher risk for developing persistent disease. In conclusion, the immunohistochemical study shows high expression of EGF in early normal placentae and moles linking its role to proliferative and differentiating activity of trophoblasts. Tumors with histological diagnosis of invasive moles and choriocarcinoma showed very strong binding of EGF and thus EGF has the potential of identifying high risk lesions.

Adolescent↗
PubMed1996

Interaction of G protein Gbetagamma dimers with small GTP-binding proteins of the Rho family.

Gbetagamma dimers of heterotrimeric G proteins have been shown to be important for the translocation of cytosolic proteins to membranes. The involvement of Gbetagamma in those signaling processes mediated by small GTP-binding proteins of the Rho family was studied using purified proteins. We showed specific binding of bovine brain Gbetagamma to immobilized GST-Rho fusion proteins. In addition, brain Gbetagamma, but not transducin Gbetagamma, was able to inhibit GTPgammaS binding to GST-Rho in a concentration-dependent manner. GTPgammaS binding to GST-Rac was also decreased by brain Gbetagamma whereas nucleotide binding to GST-Cdc42 was not changed. We conclude that Gbetagamma dimers may participate in the process of membrane attachment and/or other regulations of Rho and Rac.

Animals↗
PubMed1996

Sensitizing soluble guanylyl cyclase to become a highly CO-sensitive enzyme.

It took at least a decade to realize that the toxic gas NO is the physiological activator of soluble guanylyl cyclase (sGC), thereby acting as a signaling molecule in the nervous and cardiovascular systems. Despite its rather poor sGC-activating property, CO has also been implicated as a physiological stimulator of sGC in neurotransmission and vasorelaxation. Here, we establish YC-1 as a novel NO-independent sGC activator that potentiates both CO- and NO-induced sGC stimulation. As this potentiating effect is also observed with protoporphyrin IX which activates sGC independently of a gaseous ligand, we conclude that stabilization of the enzyme's active configuration is the underlying mechanism of YC-1's action. Moreover, the results obtained with YC-1 reveal that CO is capable of stimulating sGC to a degree similar to NO, and thus provide the molecular basis for CO functioning as a signaling molecule.

Animals↗