The ubiquitous neuropeptide galanin controls numerous functions such as endocrine secretions, intestinal motility, and behavioral activities. These regulatory effects of galanin are mediated through the interaction with specific membrane receptors and involve the pertussis toxin-sensitive guanine nucleotide binding proteins Gj/G0 as transducing elements. We report here the isolation of a cDNA coding for a human galanin receptor from a Bowes melanoma cell line cDNA expression library, by using a radioligand binding strategy. The nucleotide sequence ofthe cloned receptor reveals an open reading frame encoding a 349-amino acid protein with seven putative hydrophobic transmembrane domains and significant homology with members of the guanine nucleotide binding protein-coupled neuropeptide receptor family. The cloned receptor expressed in COS cells specifically binds human, porcine, and rat galanin with high affinity (Kd in the nanomolar range) and mediates the galanin inhibition of adenylate cyclase. A 2.8-kb galanin receptor transcript was identified in several human tissues. Cloning of this galanin receptor should enhance our knowledge of its distribution, structure, and function in human physiology and pathophysiology.Galanin, a 29-amino acid neuropeptide (30 amino acids in humans), was originally isolated from pig intestine (1) and later reported to be widely distributed in the central and peripheral nervous systems of numerous species (2). Galanin is unrelated to the other known families of regulatory peptides and, to date, remains the only known member of its own family. It exerts multiple regulatory functions such as (i) control of endocrine and exocrine pancreatic secretions, (ii) regulation of intestinal motility, and (iii) modulation of behavioral, cognitive, and sensory functions such as feeding, learning, memory, and nociception (for reviews, see refs. 2 and 3). Galanin exerts its actions via binding to specific membrane receptors (4). Biochemical and molecular studies, performed in brain and pancreas, indicate that the galanin receptor is a glycoprotein of 54 kDa (5-7) coupled to the inhibitory guanine nucleotide binding (G) protein Gi, identified as Gil, Gi2, and Gi3 in pancreatic 83 cells (8,9). Depending on the target tissue, different pathways for intracellular signaling by galanin are involved: inhibition of adenylate cyclase (10), blockage of voltage-dependent Ca2+ channels (11), and activation of ATP-sensitive K+ channels (12). Structure-activity studies, with galanin fragments (13-15) and chimeric peptides (16,17), generally emphasize the importance of the N-terminal fragment of galanin for the interaction with the peptide receptor. These studies also raise the possibility of the existence of galanin receptor subtypes, an issue that may be properly addressed with the molecular cloning of the galanin receptor.In this context, we describe here the expression cloning of a cDNA encoding a galanin receptor from the human Bowes melanoma cell line (18) (vol/vol) fetal calf serum, 6 mM glutami...
We have cloned and expressed a rat cDNA, designated GALR1-rat, that encodes a galanin receptor based on homology, pharmacology, and anatomical criteria. This cDNA was isolated from a rat brain cDNA library. The nucleotide sequence of the cloned receptor revealed an open reading frame encoding a 346-amino-acid protein, showing 90.8% identity with the previously cloned human galanin receptor. Membranes prepared from COS cells transiently expressing GALR1-rat specifically bind 125I-galanin with high affinity (Kd = 0.12 +/- 0.01 nM). Rat, porcine, and human galanin were able to displace 125I-galanin with nanomolar Ki (0.08 +/- 0.03, 0.10 +/- 0.01, and 0.14 +/- 0.03 nM, respectively), whereas the Ki values for the porcine galanin fragments galanin-(1-16), galanin-(2-29), and galanin-(3-29) were 0.95 +/- 0.21 nM, 7.14 +/- 0.51 nM, and > 1 microM, respectively. The rank order potency of these ligands is consistent with that reported for the native galanin receptor. The distribution of the mRNA corresponding to the galanin receptor encoded by GALR1-rat was determined by in situ hybridization to rat brain sections. High levels of galanin receptor mRNA were detected in the ventral hippocampal formation, thalamic, amygdala, and medulla oblongata nuclei, and in the dorsal horn of the spinal cord.
Pharmacologic gene regulation is a key technology, necessary to achieve safe, long-term gene transfer. The approaches described in the scientific literature all share in common the creation of artificial transcription factors by fusing a DNA-binding domain, a drug-binding domain and a transcription activation domain. These transcription factors activate the transgene expression upon binding of the pharmacologic agent (antibiotics of the tetracycline family, insect hormone, progesterone antagonist, or immunosuppressor drug) to the drug-binding domain. The major limitations to the use of these systems for human gene and cell therapies are the toxicity of the inducer molecule and the immunogenicity of the chimeric transcription factor. Thus, the gene regulation systems should operate with clinically approved drugs with safety records that do not conflict with the therapeutic gene expression regimen. This work focuses on the characterization of the immunogenicity of a tetracycline-activated transcription factor commonly used in preclinical gene therapy, rtTA2-M2, and its impact on reporter gene expression. We demonstrate that intramuscular injection of plasmid or adenoviral vectors encoding rtTA-M2 in outbred primates generates a cellular and humoral immune response to this transcription factor. The immune response to rtTA2-M2 blunts the duration of the expression the rtTA2-M2-controlled transgene in primates, presumably by destruction of the cells that coexpress rtTA2-M2 and the reporter or therapeutic gene. This immune response may result directly from the vectors used in this study, which prompts the development of new gene transfer vectors enabling safe and efficient pharmacologic gene regulation in clinic.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motoneurons, and has no effective treatment. Experimental studies in rodents have shown that motoneurons respond to a variety of molecules including brain-derived neurotrophic factor (BDNF). and the glial-cell line-derived neurotrophic factor (GDNF). Here we investigated the neuroprotective effect of these growth factors, encoded by an adenovirus, on the death of axotomized facial motoneurons in newborn rats. We used a new gene therapy strategy that involves gene transfer to motoneurons by intramuscular injection of an adenoviral vector, which is retrogradely transported from injected target muscle (Finiels et al.,: NeuroReport 7:373-378, 1995). A significant increased survival of motoneurons was observed in animals pretreated with adenovirus encoding BDNF (34.5%, P < 0.05) ou GDNF (41.9%, P < 0.05) 1 week after axotomy. These results indicate that pretreatment with BDNF or GDNF, using this therapeutic strategy, is able to prevent the massive death of motoneurons that normally follows axotomy in the neonatal period, opening new perspectives to limit neuronal death in degenerative disorders.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motoneurons, and has no effective treatment. Experimental studies in rodents have shown that motoneurons respond to a variety of molecules including brain-derived neurotrophic factor (BDNF), and the glial-cell line-derived neurotrophic factor (GDNF).Here we investigated the neuroprotective effect of these growth factors, encoded by an adenovirus, on the death of axotomized facial motoneurons in newborn rats. We used a new gene therapy strategy that involves gene transfer to motoneurons by intramuscular injection of an adenoviral vector, which is retrogradely transported from injected target muscle (Finiels et al.,: NeuroReport 7:373-378, 1995). A significant increased survival of motoneurons was observed in animals pretreated with adenovirus encoding BDNF (34.5%, P F 0.05) ou GDNF (41.9%, P F 0.05) 1 week after axotomy.These results indicate that pretreatment with BDNF or GDNF, using this therapeutic strategy, is able to prevent the massive death of motoneurons that normally follows axotomy in the neonatal period, opening new perspectives to limit neuronal death in degenerative disorders.
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