title: Pharmacological and biochemical characterization of a recombinant human galanin GALR1 receptor: agonist character of chimeric galanin peptides.
abstract: The galanin neuropeptide system is widely distributed throughout the brain and periphery and is thought to play a role in feeding, pain and reproduction. To evaluate the human galanin receptor 1 as a potential therapeutic target, we fully characterized its interaction with several galanin-like peptides. The human galanin receptor 1 receptor was stably expressed using an episomal system in human embryonic kidney 293E cells. Saturation isotherms using 125I-human galanin revealed two distinct populations of receptor affinity states in membranes and whole cells with picomolar and nanomolar affinities at the high- and low affinity states, respectively. A scintillation proximity assay revealed that 125I-human galanin binding in membranes reached steady-state within 2 to 2.5 hr; however, only 50% of galanin radiolabel dissociated from the receptors by excess galanin or guanosine 5'-O-3-thiotriphosphate even after 20 hr. In contrast, galanin binding in whole cells was completely reversible within 1 hr. Competition binding assays showed that galanin-like peptides bound with picomolar affinities in membranes and whole cells. These peptides behaved as full agonists as determined by the inhibition of forskolin-stimulated cyclic 3'5'-adenosine monophosphate production and the stimulation of guanosine 5'-O-(3-[35S]thiotriphosphate binding. The agonist profile of M40, a representative chimeric peptide, was found not to be the result of receptor reserve because receptor inactivation by partial alkylation experiments confirmed its full intrinsic efficacy under conditions of a "zero" reserve state. These observations suggest that the antagonist effects in vivo of M40, and perhaps other chimeric peptides, are not mediated via direct interactions with the galanin receptor 1 receptor.
authors.affiliation: CNS Diseases Research, The DuPont Pharmaceuticals Research Laboratories, Wilmington, Delaware, MD, USA.
authors.forename: L W
authors.initials: LW
authors.lastname: Fitzgerald
authors.forename: J P
authors.initials: JP
authors.lastname: Patterson
authors.forename: D S
authors.initials: DS
authors.lastname: Conklin
authors.forename: R
authors.initials: R
authors.lastname: Horlick
authors.forename: B L
authors.initials: BL
authors.lastname: Largent
mesh_terms: D000478:Alkylation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019004:Galanin / Q000494:pharmacology*; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D010446:Peptide Fragments / Q000494:pharmacology*; D011485:Protein Binding; D011869:Radioligand Assay; D044088:Receptors, Galanin; D018013:Receptors, Neuropeptide / Q000819:agonists* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D011994:Recombinant Proteins / Q000819:agonists / Q000378:metabolism
chemical_list: D007457:Iodine Radioisotopes; C079517:M40; D010446:Peptide Fragments; D044088:Receptors, Galanin; D018013:Receptors, Neuropeptide; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019004:Galanin