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Research ArticleArticle

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

Lawrence W. Fitzgerald, John P. Patterson, Deborah S. Conklin, Robert Horlick and Brian L. Largent
Journal of Pharmacology and Experimental Therapeutics November 1998, 287 (2) 448-456;
Lawrence W. Fitzgerald
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John P. Patterson
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Deborah S. Conklin
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Robert Horlick
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Brian L. Largent
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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.

Footnotes

  • Send reprint requests to: Dr. Lawrence W. Fitzgerald, CNS Diseases Research, E400/4442, The DuPont Pharmaceuticals Co., Experimental Station, P.O. Box 80400, Wilmington, DE 19880.

  • 1 Current address: Pharmacopeia, Inc., Cranbery, NJ 08512.

  • Abbreviations:
    cAMP
    cyclic 3′5′ adenosine monophosphate
    GALR
    galanin receptor
    HEK
    human embryonic kidney
    [35S]GTPγS
    guanosine 5′-O-3-[35S]thiotriphosphate
    Gpp(NH)p
    5′guanylimidodiphosphate
    SPA
    scintillation proximity assay
    PBS
    phosphate-buffered saline
    EBNA1
    Epstein Barr nuclear antigen 1
    CMV
    cytomegalovirus
    NPY
    neuropeptide Y
    PEI
    polyethyleneimine
    DMEM
    Dulbecco’s modified Eagle media, galantide or M15, galanin-(1-13)-substance P-(5-11)
    M35
    galanin-1-13)-bradykinin-(2-9)
    M40
    galanin-(1-13)-Pro-Pro-(Ala-Leu)2-Ala-NH2
    C7
    galanin-(1-13)spantide
    7TM
    seven transmembrane
    PBZ
    phenoxybenzamine
    GTP
    guanosine triphosphate
    • Received February 2, 1998.
    • Accepted June 4, 1998.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics
Vol. 287, Issue 2
1 Nov 1998
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Research ArticleArticle

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

Lawrence W. Fitzgerald, John P. Patterson, Deborah S. Conklin, Robert Horlick and Brian L. Largent
Journal of Pharmacology and Experimental Therapeutics November 1, 1998, 287 (2) 448-456;

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Research ArticleArticle

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

Lawrence W. Fitzgerald, John P. Patterson, Deborah S. Conklin, Robert Horlick and Brian L. Largent
Journal of Pharmacology and Experimental Therapeutics November 1, 1998, 287 (2) 448-456;
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