Specific binding of substance P aminoterminal heptapeptide [SP(1-7)] to mouse brain and spinal cord membranes.
Aminoterminal fragments of substance P (SP) have been previously shown to produce effects distinct, and often opposite, from those produced by the C-terminal of SP. The present investigation was initiated to determine whether N-terminal fragments interact at binding sites distinct from the neurokinin-1 (NK-1) receptor where the C-terminal sequence of SP binds with high affinity, and distinct from mu-opiate receptors, where we have previously shown the N-terminal sequence of SP to interact. A tritium-labeled aminoterminal heptapeptide of SP, 3H-SP(1-7), was synthesized, purified, and used to characterize the binding of a variety of fragments of SP and opioids in the mouse brain and spinal cord membranes. Using the reduction of SP-induced caudally directed biting and scratching behaviors as an index of biological activity, 3H-SP(1-7) was shown to be equipotent to unlabeled SP(1-7). 3H-SP(1-7) was found to bind reversibly to a saturable population of sites. Scatchard analyses of concentration-dependent saturation of binding in the brain indicated a single population of noninteracting sites with a high affinity (Kd = 2.5 nM) and a low capacity (Bmax = 29.2 fmol/mg protein). Kinetic analyses indicated an apparent dissociation equilibrium constant of 2.1 nM. Two populations of binding sites were observed in the spinal cord, one with a very high affinity (Kd = 0.03 nM) and low capacity (Bmax = 0.87 fmol/mg protein), and the other with lower affinity (Kd = 5.4 nM) and intermediate capacity (Bmax = 19.6 fmol/mg protein). Specific agonists for NK-1, NK-2, and NK-3 and delta opioid receptors, carboxyterminal fragments of SP, and a variety of other peptides did not compete at the 3H-SP(1-7) binding sites, but structurally related N-terminal peptides and (D-Ala2, NMe-Phe4, Gly-ol)-enkephalin (DAMGO) were active in displacing the ligand. The binding site for 3H-SP(1-7) appeared to be a membrane-bound complex whose specific binding was dependent on the integrity of both proteins and phospholipids. These studies are the first to characterize the binding sites for the SP N-terminal partial sequence of SP that can be generated by metabolism in vivo. The expanding body of evidence for distinct biological activities of N-terminal metabolites of SP, together with the current characterization of N-terminal binding, strongly support the existence of an N-terminal-directed SP receptor. The characteristics of SP(1-7) binding sites are consistent with those expected for an SP N-terminal receptor.
University of Minnesota, College of Veterinary Medicine, Department of Veterinary Biology, St. Paul 55108.
O J
OJ
Igwe
D C
DC
Kim
V S
VS
Seybold
A A
AA
Larson
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D007202:Indicators and Reagents; D007700:Kinetics; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D013116:Spinal Cord / Q000378:metabolism*; D013373:Substance P / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism*
D007202:Indicators and Reagents; D010446:Peptide Fragments; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter; D013373:Substance P; C036877:substance P (1-7)