TY - JOUR
T1 - Specific binding of substance P aminoterminal heptapeptide [SP(1-7)] to mouse brain and spinal cord membranes
AU - Igwe, Orisa J.
AU - Kim, Duckhee C.
AU - Seybold, Virginia S.
AU - Larson, Alice A.
PY - 1990
Y1 - 1990
N2 - 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 μ-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 δ 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.
AB - 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 μ-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 δ 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.
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U2 - 10.1523/jneurosci.10-11-03653.1990
DO - 10.1523/jneurosci.10-11-03653.1990
M3 - Article
C2 - 1700082
AN - SCOPUS:0025513648
SN - 0270-6474
VL - 10
SP - 3653
EP - 3663
JO - Journal of Neuroscience
JF - Journal of Neuroscience
IS - 11
ER -