Pharmacological and Biochemical Characterization of a Recombinant Human Galanin GALR1 Receptor: Agonist Character of Chimeric Galanin Peptides

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 pepti...

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Bibliographic Details
Published in:The Journal of pharmacology and experimental therapeutics 1998-11, Vol.287 (2), p.448-456
Main Authors: Fitzgerald, L W, Patterson, J P, Conklin, D S, Horlick, R, Largent, B L
Format: Article
Language:English
Subjects:
Alkylation
Cell Line
Cell Membrane - metabolism
Galanin - pharmacology
Humans
Iodine Radioisotopes
Kinetics
Peptide Fragments - pharmacology
Protein Binding
Radioligand Assay
Receptors, Galanin
Receptors, Neuropeptide - agonists
Receptors, Neuropeptide - metabolism
Recombinant Fusion Proteins - pharmacology
Recombinant Proteins - agonists
Recombinant Proteins - metabolism
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Summary: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 125 I-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 125 I-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-[ 35 S]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.
ISSN:0022-3565
1521-0103