A generic method for the production of cell lines expressing high levels of 7-transmembrane receptors.

G-protein-coupled or 7-transmembrane receptors (7TMRs) are often studied after heterologous expression in mammalian cells such as COS-7, CHO-K1, or HEK-293s. In this paper, we describe the development of a rapid and generic method for producing stable Chinese hamster ovary cell lines expressing high levels of recombinant 7TMRs by N-terminal tagging these proteins with the hemagglutinin (HA) sequence. To illustrate the broad applicability of this technique, we have presented data from cell lines expressing a glycoprotein hormone receptor for follicle-stimulating hormone (FSHR), CXC- (CXCR-2), and CC-chemokine (CCR-1) receptors and peptide receptors from the somatostatin (SSTR1, 2, 5) and neuropeptide Y (NPY-Y2, -Y4 Rs) families. Typically, cell lines with a receptor density of 1 to 15 pmol/mg protein are produced with this method. The presence of the HA tag does not adversely affect the binding or functional activity of the receptors.

Pharmacological characterization and selectivity of the NPY antagonist GR231118 (1229U91) for different NPY receptors.

Neuropeptide Y (NPY) is widely distributed throughout the central and peripheral nervous system and exerts a wide range of physiological responses by activating specific receptors. In this study we have characterized the potency of the high affinity peptide dimer antagonist, GR231118, to displace radiolabeled NPY/PYY from different tissues and cell lines expressing Y1 or Y2 receptors and from CHO cells stably transfected with human cDNA encoding for Y1, Y2 and Y4 receptors. GR231118 displays high affinity for Y1 and Y4 receptors, equal or better than that of NPY itself, while its activity is several fold weaker for Y2 receptors. Displacement of radiolabeled PYY from rat hypothalamic membranes by GR231118, reveals the existence of high and low affinity binding sites which may be equated to Y1 and Y2 receptors respectively suggesting that the compound maybe used as a tool to dissect central NPY receptors.

Enkephalin gene transcription in bovine chromaffin cells is regulated by calcium and protein kinase A signal transduction pathways: identification of DNase I-hypersensitive sites.

The bovine enkephalin gene is responsive to multiple signaling pathways in primary chromaffin cell cultures. We examined the effects of activation of the calcium and protein kinase A pathways on accumulation of enkephalin peptide and mRNA, gene transcription, and chromatin structure in the 5' region of the gene. We show here that the increase of enkephalin mRNA and peptide after depolarization of chromaffin cells with KCl or activation of adenylate cyclase with forskolin is preceded by an increase in enkephalin gene transcription. Both enkephalin peptide and mRNA were reduced by co-treatment of KCl- or forskolin-stimulated cultures with phorbol esters. Three enhancer sequences that were previously shown to be responsive to calcium, protein kinase A, and phorbol esters in the human gene in vitro were identified in the bovine enkephalin promoter, identifying a potential locus of control for these pathways in vivo. DNase I hypersensitivity mapping identified two tissue-specific sites that are associated with enkephalin gene expression in adrenal medulla and chromaffin cells; site 1 is in the promoter, which contains the three enhancer elements, and site 2 is in the first intron. These results suggest that regulation of the enkephalin gene in primary chromaffin cells by the calcium, protein kinase A, and protein kinase C signaling pathways occurs by modulation of transcription factor activity at several discrete loci on the enkephalin gene.

Proper glycosylation and phosphorylation of the type A natriuretic peptide receptor are required for hormone-stimulated guanylyl cyclase activity.

The natriuretic peptide receptor type A (NPR-A) is a receptor-guanylyl cyclase whose cytoplasmic enzymatic activity is stimulated by atrial natriuretic peptide binding to the extracellular domain. NPR-A expressed in COS cells is heterogeneously glycosylated, and the more highly glycosylated protein is also phosphorylated. Upon hormone binding, dephosphorylation occurs from both serine and threonine residues, probably within the kinase homology domain of NPR-A, and may be involved with receptor desensitization. Using site-specific mutations in the kinase homology domain of NPR-A, we have identified several residues that are important for regulating the guanylyl cyclase activity of NPR-A. Some of these amino acids are probably essential for maintaining the proper tertiary structure of the intracellular domain, and others may form loops that allow for binding of ATP, which is required for proper enzymatic activity. The site-specific mutants which have greatly reduced enzymatic activity are not phosphorylated and are incompletely glycosylated. These results suggest a correlation between phosphorylation and complete glycosylation of NPR-A and that both are required for hormone-induced enzymatic activity.

Hormonal induction of low affinity receptor guanylyl cyclase.

We describe a unique transient binding phenomenon for atrial natriuretic peptide (ANP) binding to the natriuretic peptide receptor-A (NPR-A) guanylyl cyclase stably expressed in 293 cells. The time course of ANP binding to intact cells peaked at 15 min followed by a subsequent decrease. Reduced binding was a consequence of an ANP induced low affinity state of NPR-A, and required the receptors' kinase homology domain. In a particulate fraction, ANP-stimulated cGMP production was dependent on ATP as a cofactor, and ATP promoted a lower affinity state. Our findings suggest that the kinase homology domain of NPR-A mediates the regulatory action of ATP, not only for signal transduction, but in the modulation of NPR-A hormone affinity.

Molecular biology of the natriuretic peptides and their receptors.

After the description in the past 5 years of BNP and CNP, interest in the natriuretic peptide family has dramatically increased. Molecular characterization of the receptors for this hormone family has identified a heterogeneity in the receptor subtypes not previously alluded to by pharmacological or biochemical studies. Much has been published on the physiology of ANP, but the major roles for BNP and CNP remain to be elucidated. Some experiments indicate that ANP and BNP may act synergistically, especially during cardiac stress; however, the high level of structural diversity of BNP among species and the ability of porcine BNP, but not human BNP, to activate human NPR-B suggest that an as yet unidentified receptor may exist that specifically recognizes BNP. Localization studies have implied that CNP is the most prominent neuropeptide in the natriuretic peptide family, and the restriction of its receptor, NPR-B, to the nervous system suggests that CNP and NPR-B may act in the brain to coordinate the central aspects of body fluid homeostasis. Of the three known NPRs, two, NPR-A and NPR-B, are capable of synthesizing their own second messenger, cGMP. The domain within these receptors that has high homology to protein kinases has been demonstrated to be essential for regulating this activity. No kinase activity has been measured in these proteins, but it is possible that this region is important for ATP regulation of guanylyl cyclase activity. This possibility raises interesting parallels with receptor-mediated cAMP signaling within cells. Seven transmembrane receptors, once activated by ligand, associate with G proteins to affect the activity of adenylyl cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

Conservation of the kinaselike regulatory domain is essential for activation of the natriuretic peptide receptor guanylyl cyclases.

The natriuretic peptide receptors, NPR-A and NPR-B, are two members of the newly described class of receptor guanylyl cyclases. The kinaselike domain of these proteins is an important regulator of the guanylyl cyclase activity. To begin to understand the molecular nature of this type of regulation, we made complete and partial deletions of the kinase domain in NPR-A and NPR-B. We also made chimeric proteins in which the kinase domains of NPR-A and NPR-B were exchanged or replaced with kinase domains from structurally similar proteins. Complete deletion of the kinase homology domain in NPR-A and NPR-B resulted in constitutive activation of the guanylyl cyclase. Various partial deletions of this region produced proteins that had no ability to activate the enzyme with or without hormone stimulation. The kinase homology domain can be exchanged between the two subtypes with no effect on regulation. However, structurally similar kinaselike domains, such as from the epidermal growth factor receptor or from the heat-stable enterotoxin receptor, another member of the receptor guanylyl cyclase family, were not able to regulate the guanylyl cyclase activity correctly. These findings suggest that the kinaselike domain of NPR-A and NPR-B requires strict sequence conservation to maintain proper regulation of their guanylyl cyclase activity.

Selective activation of the B natriuretic peptide receptor by C-type natriuretic peptide (CNP).

The natriuretic peptides are hormones that can stimulate natriuretic, diuretic, and vasorelaxant activity in vivo, presumably through the activation of two known cell surface receptor guanylyl cyclases (ANPR-A and ANPR-B). Although atrial natriuretic peptide (ANP) and, to a lesser extent, brain natriuretic peptide (BNP) are efficient activators of the ANPR-A guanylyl cyclase, neither hormone can significantly stimulate ANPR-B. A member of this hormone family, C-type natriuretic peptide (CNP), potently and selectively activated the human ANPR-B guanylyl cyclase. CNP does not increase guanosine 3',5'-monophosphate accumulation in cells expressing human ANPR-A. The affinity of CNP for ANPR-B is 50- or 500-fold higher than ANP or BNP, respectively. This ligand-receptor pair may be involved in the regulation of fluid homeostasis by the central nervous system.

Molecular cloning and expression of a receptor for human tumor necrosis factor.

A human tumor necrosis factor (TNF) binding protein from serum of cancer patients was purified to homogeneity and partially sequenced. Synthetic DNA probes based on amino acid sequence information were used to isolate cDNA clones encoding a receptor for TNF. The TNF receptor (TNF-R) is a 415 amino acid polypeptide with a single membrane-spanning region. The extracellular cysteine-rich domain of the TNF-R is homologous to the nerve growth factor receptor and the B cell activation protein Bp50. Human embryonic kidney cells transfected with a TNF-R expression vector specifically bind both 125I-labeled and biotinylated TNF-alpha. Unlabeled TNF-alpha and TNF-beta were equally effective at displacing the binding of labeled TNF-alpha to TNF-R expressing cells. Northern analysis indicates a single species of mRNA for the TNF-R in a variety of cell types. Therefore, the soluble TNF binding protein found in human serum is probably proteolytically derived from the TNF-R.

The sequence of porcine chromogranin A messenger RNA demonstrates chromogranin A can serve as the precursor for the biologically active hormone, pancreastatin.

Specific oligonucleotide priming of double-stranded DNA has been employed to sequence a porcine chromogranin A adrenomedullary cDNA. Porcine chromogranin A is more than 80% identical to human, bovine, and rat chromogranin A at its deduced N- and C-termini. A 49-amino acid region of the porcine molecule is 59-71% homologous to corresponding areas of rat, bovine, and human chromogranin A, and identical to the amino acid sequence of porcine pancreastatin. The sequence is preceded by an arginine at the N-terminus and followed by a GKR sequence at the C-terminus. Thus, porcine chromogranin A can serve as the precursor for pancreastatin, a polypeptide capable of inhibiting insulin release from the endocrine pancreas and acid secretion from parietal cells of the gut.

Thyroid hormone regulation of messenger ribonucleic acid encoding thyrotropin (TSH)-releasing hormone is independent of the pituitary gland and TSH.

Cellular levels of mRNA encoding pro TRH in the rostral paraventricular nucleus are reduced by thyroid hormones. To determine whether this regulatory effect of thyroid hormones requires a functional pituitary gland or, specifically, TSH, we examined the effect of T3 on proTRH mRNA in hypophysectomized, thyro-parathyroidectomized male rats with or without bovine TSH replacement. Hypophysectomy plus thyro-parathyroidectomy reduced serum T4 and TSH to undetectable levels in all animals and elevated TRH mRNA in the paraventricular nucleus over that of sham-operated animals. Eleven consecutive daily injections of T3 significantly reduced TRH mRNA levels in both sham controls and thyro-parathyroidectomized rats. However, 11 daily injections of bovine TSH (1 U/day) failed to alter the effect of T3 on TRH mRNA levels. These results demonstrate that the regulatory influence of thyroid hormones on the biosynthesis of TRH within the thyrotropic center of the brain is independent of the pituitary gland and of TSH.

Thyroid hormones regulate levels of thyrotropin-releasing-hormone mRNA in the paraventricular nucleus.

Cellular levels of messenger RNA encoding thyrotropin-releasing hormone (TRH) were measured in the paraventricular nucleus of the hypothalamus and the reticular nucleus of the thalamus in male rats after chemical thyroidectomy and thyroid hormone replacement. TRH mRNA levels were measured by quantitative in situ hybridization histochemistry using a 35S-labeled synthetic 48-base oligodeoxynucleotide probe and quantitative autoradiography. Chemical thyroidectomy, produced by the administration of 6-(n-propyl)-2-thiouracil (PrSur), reduced plasma thyroxine below detection limits and significantly increased TRH mRNA in the paraventricular nucleus. Treatment with exogenous L-triiodothyronine (T3) reduced TRH mRNA to the same level in both hypothyroid and euthyroid animals. Neither PrSur treatment nor T3 replacement influenced TRH mRNA levels in the reticular nucleus of the thalamus. Blot hybridization analysis of electrophoretically fractionated total RNA from pituitaries of these animals indicated that thyrotropin-beta mRNA levels were elevated after thyroidectomy and reduced by T3 treatment, showing that the pituitary-thyroid axis was indeed stimulated by PrSur treatment. These results suggest that thyroid hormones are involved, either directly or indirectly, in regulating the biosynthesis of TRH in the thyrotropic center of the hypothalamus.

Selective immunocytochemical staining of mitral cells in rat olfactory bulb with affinity purified antibodies against N-acetyl-aspartyl-glutamate.

Polyclonal antibodies were raised in rabbits against N-acetyl-aspartyl-glutamate (NAAG) coupled to bovine serum albumin (BSA) with carbodiimide and were purified by affinity chromatography sequentially over BSA-agarose and NAAG-agarose resins. Solid-phase RIA revealed a distinct pattern of specificity of the antibodies for N-acetylated acidic peptides, with highest signal obtained for NAAG, and essentially no immunoreactivity demonstrable for aspartate or glutamate. Coronal sections through carbodiimide-fixed rat olfactory bulb were incubated with the purified antiserum and antigen localization visualized by the avidin-biotin peroxidase techniques. Immunoreactivity was restricted to the mitral cells, the major excitatory projection neurons of the lateral olfactory tract, a putative glutamatergic pathway. Immunoreactivity was selectively blocked by preincubation of the antibody with 1 microgram/ml of NAAG-BSA. These results demonstrate a specific neuronal localization of NAAG-like immunoreactivity and support the candidacy of NAAG as a neurotransmitter of the lateral olfactory tract.

Use of a cDNA clone to identify a supposed precursor protein containing valosin.

Valosin, a novel 25-amino-acid peptide isolated recently from pig intestine, has several effects on the digestive system of dogs. We report here that the valosin-specific complementary DNA clone from pigs codes for a polypeptide unlike most precursors of biologically active peptides. The predicted protein lacks a characteristic amino-terminal hydrophobic signal sequence and contains no processing signals of the type acted upon by endopeptidases to generate other active peptides from precursors. Antibodies to synthetic valosin have been used to show that nearly all valosin immunoreactivity is in the cytoplasm and that the protein detected (valosin-containing protein, VCP), although smaller than the predicted product of the cDNA sequence, is much larger than valosin. Valosin-specific messenger RNA is found in extracts from many pig tissues, which contrasts with the restricted occurrence expected of a biologically active peptide. We conclude that valosin is an artefact of the purification procedure and does not occur in vivo.

The characterization of the specific binding of [3H]-N-acetylaspartylglutamate to rat brain membranes.

[3H]-N-Acetylaspartylglutamate (NAAG) bound saturably and reversibly to crude synaptosomal rat brain membranes. Optimal binding occurred in Tris-HCl buffer, pH 7.2, at 37 degrees C using previously frozen, preincubated membranes. Saturation experiments revealed an apparent KD of 383 +/- 33 nM and a Bmax of 31 +/- 2 pmol/mg of protein. [3H]NAAG specific binding was displaceable by serine-o-sulfate, quisqualate, ibotenate, and glutamate with K1's in the nanomolar range, whereas the amino-phosphono analogues displaced [3H] NAAG in the micromolar range (APB greater than APV greater than APH). No specific binding was found in peripheral tissues. Within the central nervous system, the thalamus exhibited the greatest amount of binding, whereas binding was lowest in cortex. Calcium ions enhanced the specific binding, whereas sodium ions caused a concentration-dependent inhibition. These results suggest that [3H]NAAG labels an acidic amino acid receptor site designated "A-4," which recognizes the antagonist, 2-amino-4-phosphonobutyric acid, and that this receptor may mediate the neurophysiologic effects of endogenous NAAG.

Regional brain levels of N-acetyl-aspartyl-glutamate: the effect of kindled seizures.

Rats were subjected to daily amygdaloid-kindling stimulation and sacrificed 48 h after their fifth stage-5 kindled seizure. Regional brain dissection was performed and the regions assayed for levels of N-acetyl-aspartyl-glutamate, an endogenous dipeptide specific to the brain and a putative excitatory neurotransmitter. Kindling produced significant increases in this dipeptide in the entorhinal cortex.

N-acetyl-aspartyl-glutamate: regional levels in rat brain and the effects of brain lesions as determined by a new HPLC method.

An isocratic HPLC method to measure endogenous N-acetyl-aspartyl-glutamate (NAAG) and N-acetyl-aspartate (NAA) is described. After removal of primary amines by passage of tissue extracts over AG-50 resin, the eluate was subject to HPLC anion-exchange analysis and eluted with phosphate buffer with absorbance monitored at 214 nm. The retention time for NAA was 5.6 min and for NAAG 11.4 min with a limit sensitivity of 0.1 nmol. The levels of NAA and NAAG were measured in 16 regions of rat brain and in heart and liver. NAAG was undetectable in heart and liver and exhibited 10-fold variation in concentration among brain regions; the highest levels were found in spinal cord. In contrast, low concentrations of NAA were detectable in heart and liver, and the regional distribution of NAA in brain varied only twofold. The regional distribution of NAA and NAAG correlated poorly. To assess the neuronal localization of these two compounds, the effects of selective brain lesions on their levels were examined. Decortication caused a 28% decrease in NAAG levels in the ipsilateral striatum while NAA decreased 38%. Kainate lesion of the striatum resulted in a 31% decrease in NAAG in the ipsilateral striatum, whereas NAA fell by 58%. Kainate lesion of the hippocampus resulted in significant decrements in NAAG and NAA in the hippocampus and septum. Transection of the spinal cord at midthorax resulted in a 51% decrease in NAAG levels immediately caudal and a 40% decrease immediately rostral to the lesion; however, NAA decreased only 30% in these areas. These results are consistent with a neuronal localization of NAAG in brain.(ABSTRACT TRUNCATED AT 250 WORDS)