Biomedical subjects

L R Fitzgerald

Publications and source records attributed to L R Fitzgerald.

14 recordsLinked to original sources

PubMed2000

Receptor for the pain modulatory neuropeptides FF and AF is an orphan G protein-coupled receptor.

Opiate tolerance and dependence are major clinical and social problems. The anti-opiate neuropeptides FF and AF (NPFF and NPAF) have been implicated in pain modulation as well as in opioid tolerance and may play a critical role in this process, although their mechanism of action has remained unknown. Here we describe a cDNA encoding a novel neuropeptide Y-like human orphan G protein-coupled receptor (GPCR), referred to as HLWAR77 for which NPAF and NPFF have high affinity. Cells transiently or stably expressing HLWAR77 bind and respond in a concentration-dependent manner to NPAF and NPFF and are also weakly activated by FMRF-amide (Phe-Met-Arg-Phe-amide) and a variety of related peptides. The high affinity and potency of human NPFF and human NPAF for HLWAR77 strongly suggest that these are the cognate ligands for this receptor. Expression of HLWAR77 was demonstrated in brain regions associated with opiate activity, consistent with the pain-modulating activity of these peptides, whereas the expression in adipose tissue suggests other physiological and pathophysiological activities for FMRF-amide neuropeptides. The discovery that the anti-opiate neuropeptides are the endogenous ligands for HLWAR77 will aid in defining the physiological role(s) of these ligands and facilitate the identification of receptor agonists and antagonists.

Amino Acid Sequence↗

PubMed2000

Neuromedin U is a potent agonist at the orphan G protein-coupled receptor FM3.

Neuromedins are a family of peptides best known for their contractile activity on smooth muscle preparations. The biological mechanism of action of neuromedin U remains unknown, despite the fact that the peptide was first isolated in 1985. Here we show that neuromedin U potently activates the orphan G protein-coupled receptor FM3, with subnanomolar potency, when FM3 is transiently expressed in human HEK-293 cells. Neuromedins B, C, K, and N are all inactive at this receptor. Quantitative reverse transcriptase-polymerase chain reaction analysis of neuromedin U expression in a range of human tissues showed that the peptide is highly expressed in the intestine, pituitary, and bone marrow, with lower levels of expression seen in stomach, adipose tissue, lymphocytes, spleen, and the cortex. Similar analysis of FM3 expression showed that the receptor is widely expressed in human tissue with highest levels seen in adipose tissue, intestine, spleen, and lymphocytes, suggesting that neuromedin U may have a wide range of presently undetermined physiological effects. The discovery that neuromedin U is an endogenous agonist for FM3 will significantly aid the study of the full physiological role of this peptide.

Calcium↗

PubMed1999

Measurement of responses from Gi-, Gs-, or Gq-coupled receptors by a multiple response element/cAMP response element-directed reporter assay.

We have established a rapid, sensitive, high-throughput assay that requires one assay condition to detect agonist effects from Gi-, Gs-, and Gq-coupled receptors. We utilized a vector containing a promoter with three multiple response elements, the vasoactive intestinal peptide promoter and a cAMP response element controlling the transcription of the luciferase gene. An adrenergic agonist, para-aminoclonidine, inhibited forskolin-stimulated luciferase expression when cells were cotransfected with the Gi-coupled alpha(2)-C adrenergic receptor and the MRE/CRE reporter vector. Further, we demonstrate that gastrin-releasing peptide, which activates a Gq-coupled GRP receptor, isoproterenol, which activates a Gs-coupled beta-adrenergic receptor, calcium ionophores, and phorbol 12-myristate 13-acetate, a stimulator of protein kinase C, can mediate increases in luciferase expression in the presence of forskolin but not in its absence. The effect at Gi-coupled receptor activation correlates with the phosphorylation of the CRE binding protein (CREB); however, the mechanisms mediating the responses to Gq- and Gs-coupled receptors are more complex. We demonstrate that this assay is useful for pharmacological analysis of both agonists and antagonists and has the potential to associate orphan G-protein-coupled receptors with their corresponding ligands.

Base Sequence↗

PubMed1999

Melanin-concentrating hormone is the cognate ligand for the orphan G-protein-coupled receptor SLC-1.

The underlying causes of obesity are poorly understood but probably involve complex interactions between many neurotransmitter and neuropeptide systems involved in the regulation of food intake and energy balance. Three pieces of evidence indicate that the neuropeptide melanin-concentrating hormone (MCH) is an important component of this system. First, MCH stimulates feeding when injected directly into rat brains; second, the messenger RNA for the MCH precursor is upregulated in the hypothalamus of genetically obese mice and in fasted animals; and third, mice lacking MCH eat less and are lean. MCH antagonists might, therefore, provide a treatment for obesity. However, the development of such molecules has been hampered because the identity of the MCH receptor has been unknown until now. Here we show that the 353-amino-acid human orphan G-protein-coupled receptor SLC-1 expressed in HEK293 cells binds MCH with sub-nanomolar affinity, and is stimulated by MCH to mobilize intracellular Ca2+ and reduce forskolin-elevated cyclic AMP levels. We also show that SLC-1 messenger RNA and protein is expressed in the ventromedial and dorsomedial nuclei of the hypothalamus, consistent with a role for SLC-1 in mediating the effects of MCH on feeding.

Alternative Splicing↗

PubMed1997

PTP NE-6: a brain-enriched receptor-type protein tyrosine phosphatase with a divergent catalytic domain.

A receptor-type protein tyrosine phosphatase, PTP NE-6, was identified from rat olfactory epithelial cDNA and cloned from a rat brain cDNA library. PTP NE-6 mRNA is abundant in brain and expressed at lower levels in olfactory tissue and adrenal gland. In situ hybridization demonstrates that PTP NE-6 mRNA is expressed throughout the brain, with highest levels in the medial habenula and at intermediate levels in layer IV of cortex, medial geniculate nucleus, inferior colliculus, hypothalamus, and thalamus. The predicted amino acid sequence demonstrates that PTP NE-6 contains a single catalytic domain that diverges from the consensus protein tyrosine phosphatase catalytic domain by expressing an aspartate instead of the conserved alanine residue in the catalytic site. Recombinantly expressed PTP NE-6 does not exhibit detectable phosphatase activity. Upon mutation of the aspartate to the consensus alanine, phosphatase activity toward p-nitrophenyl phosphate is observable with a k(cat) value of 3.7 s(-1) and a Km of 980 microM. These data demonstrate that the inactivity of native PTP NE-6 toward p-nitrophenyl phosphate is due to the divergent aspartate in the catalytic site and not to variant amino acids within the phosphatase domain.

Amino Acid Sequence↗

PubMed1996

Electroconvulsive seizure increases the expression of CREM (cyclic AMP response element modulator) and ICER (inducible cyclic AMP early repressor) in rat brain.

Rapid expression of ICER (inducible cyclic AMP early repressor), an inducible member of the CREM (cyclic AMP response element modulator) family of transcription factors, has been reported in neuroendocrine tissues and cell lines, but not in brain. In the present study, we demonstrate that acute electro-convulsive seizure (ECS) increases the expression of ICER in several rat brain regions. RNase protection analysis demonstrated that 1-2 h after administration of ECS, levels of mRNA for ICER and a splice variant, ICER gamma, were significantly increased in hippocampus, frontal cortex, and cerebellum. It is surprising that ECS also increased levels of mRNA for several CREM isoforms that previous studies have reported were not rapidly inducible. In situ hybridization analysis confirmed these findings and demonstrated that ECS induction of ICER was most obvious in the dentate gyrus granule cell layer of hippocampus and deep layers of cerebral cortex. Induction of ICER and CREM was accompanied by increased expression of two small CRE-binding complexes. Gel supershift analysis with CREM/ICER antisera confirmed that the inducible CRE-binding complexes contain CREM/ICER. Induction of CREM and ICER may contribute to negative feedback regulation of gene transcription that is increased by acute seizure and activation of CREB (cyclic AMP response element-binding protein.

Animals↗

PubMed1996

Adrenergic regulation of ICER (inducible cyclic AMP early repressor) and beta1-adrenergic receptor gene expression in C6 glioma cells.

ICER (inducible cyclic AMP early repressor), a member of the cyclic AMP response element (CRE) modulator (CREM) family of transcription factors, is a powerful repressor of cyclic AMP-mediated transactivation. Our studies characterize the regulation of ICER in C6 glioma cells and investigate its role in repressing transcription of the beta1-adrenergic receptor (beta1AR) gene. Incubation with isoproterenol (100 nM) results in a rapid induction in levels of mRNA for ICER and its splice variant ICERgamma, with maximal induction occurring after 2 h of treatment. Incubation with isoproterenol also increased levels of CREM isoforms within 1 h; this was unexpected given previous reports that these isoforms are not rapidly induced. Increased expression of ICER and CREM was accompanied by induction of two CRE-binding complexes. The presence of ICER in these two CRE-binding complexes is demonstrated by their disruption with CREM antibody and by their comigration with recombinant ICER. Because the time course for isoproterenol induction of ICER mRNA and CRE binding corresponds to that for down-regulation of beta1AR mRNA levels in C6 glioma cells, the influence of ICER beta1AR transcription was directly examined. Coexpression of ICER significantly decreased transcriptional activity of a rat beta1AR promoter-luciferase reporter construct that contains a CRE. In contrast, coexpression of ICER did not influence two truncated rat beta1AR promoter constructs that lack the CRE site. These data demonstrate that ICER can interact at the beta1AR promoter to repress transcription.

Animals↗

PubMed1995

Regulation of beta 2-adrenergic receptor mRNA and gene transcription in rat C6 glioma cells: effects of agonist, forskolin, and protein synthesis inhibition.

Incubation of rat C6 glioma cells with beta-adrenergic receptor (beta AR) agonist or with agents that increase cAMP levels results in down-regulation of the beta 2AR, as measured by the loss of radioligand binding sites. In the present study, the role of beta 2AR mRNA expression and stability in the down-regulation of beta 2AR sites in C6 cells was examined. Isoproterenol or forskolin treatment decreased beta 2AR mRNA levels in a time-dependent manner, with maximal loss of approximately 50% being observed after 2 hr. Pretreatment of the cells with a potent protein synthesis inhibitor, Pseudomonas exotoxin A, completely blocked isoproterenol- and forskolin-mediated down-regulation of beta 2AR mRNA. Exposure to agonist did not significantly influence the half-life of beta 2AR mRNA, which was approximately 60 min. In contrast, isoproterenol treatment for 2 hr significantly decreased the rate of beta 2AR gene transcription, as determined by nuclear run-on analysis. Based on these results, we propose that agonist regulation of beta 2AR mRNA in C6 cells is mediated by activation of the cAMP system and occurs at the level of beta 2AR gene transcription, not mRNA stability. In addition, the observed requirement for protein synthesis indicates that down-regulation of beta 2AR mRNA may be mediated by expression of a repressor of beta 2AR gene transcription.

ADP Ribose Transferases↗

PubMed1989

Effect of striatal implantation of bovine adrenal chromaffin cells on turning behavior in a rat model of Parkinson's disease.

Rats lesioned unilaterally with 6-hydroxydopamine, as an animal model of Parkinson's disease, were tested for D-amphetamine-induced rotation. Buffer or isolated bovine adrenal chromaffin cells were implanted into the lesioned striatum. Rats were retested for rotation 1 and 4 weeks postimplantation. Those with surviving implants rotated significantly less than controls. HPLC assay of striata showed that implants contained elevated norepinephrine and epinephrine, but not dopamine. The results show that the bovine chromaffin cell implants can function as a restorative treatment for nigrostriatal damage, that neonatal implants work better than adult implants, and that such restoration occurs independent of dopamine levels.

Adrenal Glands↗

PubMed1983