Regulation of human neuronal calcium channels by G protein betagamma subunits expressed in human embryonic kidney 293 cells

Serotonin (5-HT) was found to inhibit steroid (17 alpha,20 beta-dihydroxy-4-pregnen-3-one; 17,20 beta P)-induced resumption of oocyte meiosis (oocyte maturation) in vitro in the teleost Fundulus heteroclitus. Serotonin inhibited both follicle-enclosed and denuded oocytes, which indicates the presence of oocyte-associated 5-HT sensitive sites. The response of oocytes to 5-HT was characterized pharmacologically, i.e., the capacity of serotonergic agonists and antagonists to mimic or block the 5-HT inhibition of the steroid-induced oocyte maturation was assessed by the changes in the percentage of oocyte germinal vesicle breakdown (GVBD). Dose-response curves for each compound were drawn and compared. The rank order of potency among the agonists was: 5-HT > 5-methoxytryptamine > tryptamine = 5,6-diHT = 5-carboxidotryptamine > 5,7-diHT = 5-methoxy-dimethyltryptamine > alpha-methyl-5HT > 2-methyl-5HT. Incubation of ovarian follicles with high doses of some antagonists (mianserin and metergoline) induced oocyte GVBD, although this effect was associated with high levels of oocyte atresia during GVBD or shortly after maturation. Consequently, doses of the antagonist too low to induce GVBD were tested for their ability to block the 5-HT inhibitory action; the rank order of potency was: MDL-72222 = metoclopramide > metergoline > propanolol > ketanserin. Dopamine, acetylcholine, epinephrine, and norepinephrine could also inhibit 17,20 beta P-induced GVBD, although at doses much higher than those of 5-HT; melatonin and histamine had no effect on oocyte maturation. These results suggest that specific receptors mediate the inhibitory action of 5-HT on the steroid-triggered meiosis resumption. The pharmacological profile of these 5-HT receptors is different from those of any known mammalian 5-HT receptor, although they showed some similarities to the 5-HT1A, 5-HT2, and 5-HT3 receptors, as well as to 5-HT receptors on oocytes of some bivalve molluscs.     

Characterization of insulin receptor substrate 4 in human embryonic kidney 293 cells

We recently cloned IRS-4, a new member of the insulin receptor substrate (IRS) family. In this study we have characterized IRS-4 in human embryonic kidney 293 cells, where it was originally discovered. IRS-4 was the predominant insulin-elicited phosphotyrosine protein in these cells. Subcellular fractionation revealed that about 50% of IRS-4 was located in cellular membranes, and immunofluorescence indicated that IRS-4 was concentrated at the plasma membrane. Immunoelectron microscopy conclusively established that a large portion of the IRS-4 was located at the cytoplasmic surface of the plasma membrane in both the unstimulated and insulin-treated states. IRS-4 was found to be associated with two src homology 2 (SH2) domain-containing proteins, phosphatidylinositol 3-kinase and Grb2, the adaptor to the guanine nucleotide exchange factor for Ras. On the other hand, no significant association was detected with two other SH2 domain proteins, the SH2-containing protein tyrosine phosphatase 2 and phospholipase Cgamma. Insulin-like growth factor I acting through its receptor was as effective as insulin in eliciting tyrosine phosphorylation of IRS-4, but interleukin 4 and epidermal growth factor were ineffective.     

Agonist-dependent internalization of the human melanocortin-4 receptors in human embryonic kidney 293 cells

A chimeric protein comprised of melanocortin-4 receptor (MC4R) and the green fluorescent protein (GFP) was created for studying receptor/ligand localization and trafficking. The ligand binding affinities and second messenger stimulation induced by MC4R-GFP closely resembled those of the wild-type receptor, suggesting functional integrity of the chimeric protein. As observed with a confocal microscope, in human embryonic kidney (HEK)-293 cells MC4R/GFP was distributed evenly along the cell membrane. Addition of [Nle4-d-Phe7]-alpha-melanocyte-stimulating hormone (NDP-MSH), a peptide MC4R agonist, induced receptor translocation into intracellular compartments in a time- and concentration-dependent manner. [Ac-Nle-c[Asp-His-d-Nal(2')-Arg-Trp-Lys]-NH2] (SHU9119), a potent MC4R antagonist, completely inhibited NDP-MSH-mediated internalization. MC4R-GFP internalization was unaffected by a protein kinase A inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), but was impaired by pretreatment with inhibitors of endocytosis through clathrin-coated pits, hypertonic sucrose, or concanavalin A. Time-dependent colocalization of MC4R-GFP with rhodamine-transferrin, an early endosome marker, and with LysoTraker, a lysosome marker, was observed after short-term (45 min) and prolonged (20 h) agonist exposure, respectively. Rhodamine-[AcNle-c[Asp-His-d-Phe-Arg-Trp-Lys]-NH2] (MTII), a fluorescent derivative of an MC4R agonist, was found to cointernalize with MC4R-GFP into intracellular vesicles. No significant receptor recycling or segregation from the ligand was observed 60 min after removal of the agonist. In contrast, an antagonist rhodamine-Ac-Cys-Glu-His-(d-Nal)-Arg-Trp-Gly-Cys-Pro-Pro-Lys-Asp-NH2 (HS014) bound to and colocalized with MC4R-GFP on the cell surface and did not stimulate receptor internalization. In sum, these results suggest that MC4R is subject to agonist-dependent endocytosis via clathrin-coated pits. Prolonged agonist exposure directs MC4R into lysosomes, possibly for degradation. Receptor and ligand recycling is not efficient for MC4R in HEK-293 cells.     

Receptor-activated Ca2+ influx via human Trp3 stably expressed in human embryonic kidney (HEK)293 cells. Evidence for a non-capacitative Ca2+ entry

Gonadal differentiation involves a complex interplay of developmental pathways. The sex determining region Y (SRY) gene plays a key role in testis determination, but its interaction with other genes is less well understood. Abnormalities of gonadal differentiation result in a range of clinical problems. 46,XY complete gonadal dysgenesis is defined by an absence of testis determination. Subjects have female external genitalia and come to clinical attention because of delayed puberty. Individuals with 46,XY partial gonadal dysgenesis usually present in the newborn period for the valuation of ambiguous genitalia. Gonadal histology always shows an abnormality of seminiferous tubule formation. A diagnosis of 46,XY true hermaphroditism is made if the gonads contain well-formed testicular and ovarian elements. Despite the pivotal role of the SRY gene in testis development, mutations of SRY are unusual in subjects with a 46,XY karyotype and abnormal gonadal development. 46,XX maleness is defined by testis determination in an individual with a 46,XX karyotype. Most affected individuals have a phenotype similar to that of Klinefelter syndrome. In contrast, subjects with 46,XX true hermaphroditism usually present with ambiguous genitalia. The majority of subjects with 46,XX maleness have Y sequences including SRY in genomic DNA. However, only rare subjects with 46,XX true hermaphroditism have translocated sequences encoding SRY. Mosaicism and chimaerism involving the Y chromosome can also be associated with abnormal gonadal development. However, the vast majority of subjects with 45,X/46,XY mosaicism have normal testes and normal male external genitalia.     

FasL induces Fas/Apo1-mediated apoptosis in human embryonic kidney 293 cells routinely used to generate E1-deleted adenoviral vectors

Phosphatidylinositol-4-phosphate 5-kinase (PIP5K) phosphorylates phosphatidylinositol-4-phosphate to produce phosphatidylinositol-4,5-bisphosphate as a precursor of two second messengers, inositol-1,4,5-triphosphate and diacylglycerol, and as a regulator of many cellular proteins involved in signal transduction and cytoskeletal organization. Despite PIP5K playing such an essential role in a number of physiological processes, much still remains to be made clear about its association with plants. Searching the Arabidopsis expression sequence tag database against already known yeast and mammalian PIP5K cDNAs, we identified two clones which partly encode the same Arabidopsis PIP5K and isolated a corresponding full-length cDNA encoding a protein that we designated AtPIP5K1. Recombinant AtPIP5K1 expressed in Escherichia coli possessed a PIP5K activity in vitro. Due to some structural and biochemical differences, AtPIP5K1 was not categorized as either a type I or type II PIP5K. The expression of the AtPIP5K1 mRNA was induced rapidly by treating Arabidopsis plants with drought, salt and abscisic acid, which suggests that AtPIP5K11 is involved in water-stress signal transduction. These data give evidence for a close link between phosphoinositide signaling cascades and water-stress responses in plants.     

Differential localization of vesicular acetylcholine and monoamine transporters in PC12 cells but not CHO cells

Previous studies have indicated that neuro-endocrine cells store monoamines and acetylcholine (ACh) in different secretory vesicles, suggesting that the transport proteins responsible for packaging these neurotransmitters sort to distinct vesicular compartments. Molecular cloning has recently demonstrated that the vesicular transporters for monoamines and ACh show strong sequence similarity, and studies of the vesicular monoamine transporters (VMATs) indicate preferential localization to large dense core vesicles (LDCVs) rather than synaptic-like microvesicles (SLMVs) in rat pheochromocytoma PC12 cells. We now report the localization of the closely related vesicular ACh transporter (VAChT). In PC12 cells, VAChT differs from the VMATs by immunofluorescence and fractionates almost exclusively to SLMVs and endosomes by equilibrium sedimentation. Immunoisolation further demonstrates colocalization with synaptophysin on SLMVs as well as other compartments. However, small amounts of VAChT also occur on LDCVs. Thus, VAChT differs in localization from the VMATs, which sort predominantly to LDCVs. In addition, we demonstrate ACh transport activity in stable PC12 transformants overexpressing VAChT. Since previous work has suggested that VAChT expression confers little if any transport activity in non-neural cells, we also determined its localization in transfected CHO fibroblasts. In CHO cells, VAChT localizes to the same endosomal compartment as the VMATs by immunofluorescence, density gradient fractionation, and immunoisolation with an antibody to the transferrin receptor. We have also detected ACh transport activity in the transfected CHO cells, indicating that localization to SLMVs is not required for function. In summary, VAChT differs in localization from the VMATs in PC12 cells but not CHO cells.     

Characterization of human type X procollagen and its NC-1 domain expressed as recombinant proteins in HEK293 cells

Type X collagen is a short-chain, network-forming collagen found in hypertrophic cartilage in the growth zones of long bones, vertebrae, and ribs. To obtain information about the structure and assembly of mammalian type X collagen, we generated recombinant human type collagen X by stable expression of full-length human alpha1(X) cDNA in the human embryonal kidney cell line HEK293 and the fibrosarcoma cell line HT1080. Stable clones were obtained secreting recombinant human type X collagen (hrColX) in amounts of 50 microg/ml with alpha1(X)-chains of apparent molecular mass of 75 kDa. Pepsin digestion converted the native protein to a molecule migrating as one band at 65 kDa, while bands of 55 and 43 kDa were generated by trypsin digestion. Polyclonal antibodies prepared against purified hrColX reacted specifically with type X collagen in sections of human fetal growth cartilage. Circular dichroism spectra and trypsin/chymotrypsin digestion experiments of hrColX at increasing temperatures indicated triple helical molecules with a reduced melting temperature of 31 degrees C as a result of partial underhydroxylation. Ultrastructural analysis of hrColX by rotary shadowing demonstrated rodlike molecules with a length of 130 nm, assembling into aggregates via the globular noncollagenous (NC)-1 domains as reported for chick type X collagen. NC-1 domains generated by collagenase digestion of hrColX migrated as multimers of apparent mass of 40 kDa on SDS-polyacrylamide gel electrophoresis, even after reduction and heat denaturation, and gave rise to monomers of 18-20 kDa after treatment with trichloroacetic acid. The NC-1 domains prepared by collagenase digestion comigrated with NC-1 domains prepared as recombinant protein in HEK293 cells, both in the multimeric and monomeric form. These studies demonstrate the potential of the pCMVsis expression system to produce recombinant triple helical type X collagens in amounts sufficient for further studies on its structural and functional domains.     

Studies of melatonin effects on epithelia using the human embryonic kidney-293 (HEK-293) cell line

The expression of melatonin receptors (MR) of the Mel1a subtype in basolateral membrane of guinea pig kidney proximal tubule suggests that melatonin plays a role in regulating epithelial functions. To investigate the cellular basis of melatonin action on epithelia, we sought to establish an appropriate in vitro culture model. Epithelial cell lines originating from kidneys of dog (MDCK), pig (LLC-PK1), opossum (OK), and human embryo (HEK-293) were each tested for the presence of MR using 2-[125I]iodomelatonin (125I-MEL) as a radioligand. The HEK-293 cell line exhibited the highest specific 125I-MEL binding. By intermediate filament characterization, the HEK-293 cells were determined to be of epithelial origin. Binding of 125I-MEL in HEK-293 cells demonstrated saturability, reversibility, and high specificity with an equilibrium dissociation constant (Kd) value of 23.8 +/- 0.5 pM and a maximum number of binding sites (Bmax) value of 1.17 +/- 0.11 fmol/mg protein (n = 5), which are comparable with the reported Kd and Bmax values in human kidney cortex. Coincubation with GTPgammaS (10 microM) and pertussis toxin (100 ng/ml) provoked a marked decrease in binding affinity (Kd was increased by a factor of 1.5-2.0), with no significant difference in Bmax. Melatonin (1 microM) decreased the forskolin (10 microM) stimulated cAMP level by 50%. HEK-293 cells do not express dopamine D1A receptor. Following transient transfection of HEK-293 cells with human dopamine D1A receptor (hD1A-R), exposure of the cells to dopamine stimulated an increase in the level of cAMP. Similarly, transient transfection of HEK-293 cells with rat glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), and PTH type 1 receptors, each resulted in an hormone inducible increase in cAMP levels. Surprisingly, only the stimulatory effect of dopamine could be inhibited by exposure to melatonin. The inhibitory effect of melatonin on dopamine D1-induced increase in cAMP was completely inhibited by pertussis toxin (100 ng/ml, 18 h). Immunoblot and immunocytochemical studies were carried out using two polyclonal antibodies raised against the extra and cytoplasmic domains of Mel1a receptor. Immunoblot studies using antibody against the cytoplasmic domain of Mel1a receptor confirmed the presence of a peptide blockable 37 kDa band in HEK-293 cells. Indirect immunofluorescent studies with both antibodies revealed staining predominantly at the cell surface, but staining with the antibody directed against the cytoplasmic domain required prior cell permeabilization. By RT-PCR, HEK-293 cells express both Mel1a and Mel1b messenger RNAs, but the messenger RNA level for Mel1b is several orders of magnitude lower than for Mel1a. We conclude that HEK-293 cells express MR predominantly of the Mel1a subtype. Our evidence suggests that one of the ways that melatonin exerts its biological function is through modulation of cellular dopaminergic responses.     

Pituitary cell line GH3 expresses two somatostatin receptor subtypes that inhibit adenylyl cyclase: functional expression of rat somatostatin receptor subtypes 1 and 2 in human embryonic kidney 293 cells

Using a polymerase chain reaction approach, we have studied the expression of somatostatin receptor (SSTR) subtypes in the GH3 rat pituitary cell line, a well established in vitro model for the cellular effects of somatostatin. We found that the previously identified SSTR1 and SSTR2 are the major subtypes expressed in this cell line. No other SSTR subtype was detected by our analysis. Northern blots confirmed that both subtypes, but not SSTR3, are expressed in GH3 cells. We studied the functional expression of both SSTR subtypes by transfection of their cDNAs into human embryonic kidney 293 cells. We found that somatostatin inhibited cAMP accumulation in human embryonic kidney 293 cells only when cells were transfected with either SSTR1 or SSTR2. This inhibition was blocked by treatment of the transfected cells with pertussis toxin, demonstrating that it is mediated by G proteins sensitive to this toxin. In addition, we provide pharmacological evidence that the endogenous SSTR2 subtype mediates inhibition of cAMP accumulation in intact GH3 cells. Our results contradict previous reports that concluded thsat neither SSTR1 nor SSTR2 is involved in inhibition of adenylyl cyclase. The reasons for this apparent contradiction are discussed. We conclude that both SSTR1 and SSTR2 are capable of coupling to pertussis toxin-sensitive G proteins to inhibit adenylyl cyclase.     

The human 5-ht5A receptor couples to Gi/Go proteins and inhibits adenylate cyclase in HEK 293 cells

The G protein coupling of human 5-hydroxytryptamine5A (h5-ht5A) receptors was investigated in stably transfected human embryonic kidney (HEK) 293 cells, using radioligand and guanosine-5'[gamma-35S]thiotriphosphate binding to membranes and cyclic adenosine monophosphate measurements in cells. 5-Carboxamido[3H]tryptamine bound to high- and low-affinity sites on h5-ht5A-HEK 293 cell membranes. Guanylyl-imidodiphosphate addition and pertussis toxin pre-treatment abolished high-affinity binding, indicating coupling to G proteins of the Gi/Go family. [N-methyl-3H]Lysergic acid diethylamide bound to a single site; guanylyl-imidodiphosphate and pertussis toxin did not alter lysergic acid diethylamide affinity. 5-Hydroxytryptamine stimulated guanosine-5'[gamma-35S]thiotriphosphate binding to 130% over basal and this effect was completely abolished by pertussis toxin. Various 5-hydroxytryptamine receptor ligands were tested for inhibition of 5-carboxamido[3H]tryptamine binding and in guanosine-5'[gamma-35S]thiotriphosphate binding assays. 5-Hydroxytryptamine consistently inhibited forskolin-induced cyclic adenosine monophosphate formation by 25% in h5-ht5A-HEK 293 cells; no effect was detected on basal cyclic adenosine monophosphate levels, on intracellular Ca2+ concentration or arachidonic acid release. Our studies demonstrate functional coupling of the h5-ht5A receptor to pertussis toxin-sensitive G proteins and to inhibition of adenylate cyclase activity.     

PET studies on brain monoamine transporters with carbon-11-beta-CIT in Parkinson's disease

The cocaine analog 2 beta-carbomethoxy-3 beta-[4-iodophenyl]tropane (beta-CIT) labeled with 11C was used to study dopamine reuptake sites with PET. METHODS: Three normal subjects and nine patients with Parkinson's disease were investigated. Each of them underwent a dynamic PET scan (25 timeframes over 80 min) with [11C]-beta-CIT. A dose of 102.5-211.3 MBq (2.77-5.71 mCi) of this ligand was administered intravenously and a PET examination with an ECAT 931/08 PET camera was carried out. Ratios between the striatal/cortical/thalamic/midbrain and cerebellar uptake of this radioligand were calculated. RESULTS: The highest accumulation of [11C]beta-CIT was observed in the caudate and putamen, though there was some uptake in the thalamus and the midbrain. Cortical uptake was negligible. Carbon-11-beta-CIT accumulated significantly less in the putamen of the Parkinson's patients than in the normal subjects. The putamen-to-cerebellum ratio in the Parkinson's patients was 1.59 +/- 0.04 and 1.80 +/- 0.13s (p = 0.028) in the normal subjects. In the caudate, there was no significant difference between the Parkinson's patients and the normal subjects. CONCLUSION: These results imply that [11C]beta-CIT is a useful compound for carrying out a PET examination of the function of the presynaptic monoaminergic neurons both in normal and pathological brains.     

Down-regulation of the human norepinephrine transporter in intact 293-hNET cells exposed to desipramine

The effects of continuous exposure of cultured cells expressing the human norepinephrine transporter (hNET) to the hNET inhibitor desipramine on hNET expression and function were studied. Exposure of HEK-293 cells transfected stably with the hNET cDNA (293-hNET cells) to desipramine for 3 days reduced the specific binding of [3H]nisoxetine in membrane homogenates in a concentration-dependent manner. The magnitude of the reductions in [3H]nisoxetine binding to hNET was dependent on the length of time of the exposure to desipramine, reaching 77% after a 21-day exposure. The reduction of [3H]nisoxetine binding returned to control levels within 72 h after a 3-day exposure to desipramine. Reductions in [3H]nisoxetine binding to hNET were accompanied by time-dependent and exposure concentration-dependent reductions in hNET protein levels as determined by western blotting. Similar to binding, hNET protein levels returned to control levels 72 h after cessation of desipramine exposure. Northern blotting indicated that exposure of 293-hNET cells to desipramine did not significantly alter hNET mRNA levels. Uptake of [3H]norepinephrine by 293-hNET cells was markedly reduced after a 3-day exposure to desipramine. However, desipramine exposure had no effect on uptake of [3H]glutamate or [3H]alanine. The present findings imply that down-regulation of the hNET in 293-hNET cells induced by desipramine results from a selective reduction in hNET protein levels, presumably a consequence of either a reduction in the translation of hNET mRNA or from an enhanced degradation of hNET protein.