The three- dimensional structure of the binding pockets of the SP and SK receptors will be defined with respect to their transmembrane alpha-helices and extracellular domains by determining the sites of covalent attachment of a series of SP and SK derivatives containing the photoreactive amino acid p-benzoylphenylalanine spaced at different positions along the amino acid sequences of the two peptides. For each photoaffinity ligand, the amino acid residue of the binding site that becomes covalently labelled will be determined by microsequencing of purified receptor fragments initially located in the primary sequence with the aid of site-specific antibodies and mass spectrometry. A comparative analysis of the similar and distinctive features of these binding domains should provide insight into the molecular basis of peptide selectivity and receptor activation. A related project will be to determine the site of attachment of a tritiated photoreactive azide derivative of the nonpeptide SP antagonist CP-96,345. An understanding of the relationship between the sites for peptide agonists and the nonpeptide antagonist may provide insight into the mechanism of the antagonist effects of this compound, and the information to be gained from the project as a whole may advance the rational design of therapeutic agents for clinical entities in which these peptides participate.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS031346-01
Application #
3418265
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1992-12-01
Project End
1996-11-30
Budget Start
1992-12-01
Budget End
1993-11-30
Support Year
1
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Boston University
Department
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
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Bremer, A A; Tansky, M F; Wu, M et al. (2001) Direct evidence for the interaction of neurokinin A with the tachykinin NK(1) receptor in tissue. Eur J Pharmacol 423:143-7
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Pellegrini, M; Bremer, A A; Ulfers, A L et al. (2001) Molecular characterization of the substance P*neurokinin-1 receptor complex: development of an experimentally based model. J Biol Chem 276:22862-7
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Li, H; Hsu, P; Sachais, B S et al. (1996) Identification of the site in the substance P (NK-1) receptor for modulation of peptide binding by sulfhydryl reagents. J Biol Chem 271:1950-6
MacDonald, D; Silberman, S C; Lowe 3rd, J A et al. (1996) Photoaffinity labeling of the human substance P (neurokinin-1) receptor with [3H2]azido-CP-96,345, a photoreactive derivative of a nonpeptide antagonist. Mol Pharmacol 49:808-13
Macdonald, S G; Dumas, J J; Boyd, N D (1996) Chemical cross-linking of the substance P (NK-1) receptor to the alpha subunits of the G proteins Gq and G11. Biochemistry 35:2909-16