Photoaffinity Labeling of the Two Forms of Serotonin Binding Protein: Peptide Mapping of the Binding Sites

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Abstract
Serotonin binding protein (SBP) is a vesicular protein found in neurectoderm‐derived cells that store 5‐hydroxytryptamine (5‐HT, serotonin), such as central and peripheral serotonergic neurons and paraneurons (parafollicular cells of the thyroid). 5‐HT is stored as a complex with SBP in vivo. Two forms of the protein are found. These differ in molecular mass: one is 45 kDa and the other 56 kDa. It has been suggested that the 56‐kDa form of SBP may be the precursor of the 45‐kDa form. To study the relationship between these two proteins, we have used a covalently bound radiolabeled probe to analyze their binding domains. A photoaffinity reagent, N‐(4‐azido‐2‐nitrophenyl)‐5‐hydroxytryptamine (NAP‐5‐HT), was synthesized and characterized by nuclear magnetic resonance spectroscopy, mass spectra, and UV‐visible absorption spectra. A 1 M excess of NAP‐5‐HT inhibited the binding of [3H]5‐HT to SBP by 50%. NAP[3H]5‐HT was also synthesized and attached to both high‐ and lowaffinity binding sites on both forms of SBP. The high‐affinity binding constants for 45‐kDa and 56‐kDa proteins were 0.8 nM and 0.02 nM, respectively, whereas the low‐affinity constants were 0.3 γM and 0.15 γM. When the high‐affinity site of partially purified SBP was photoaffinity‐labeled with the reagent, two covalently labeled proteins (45 kDa and 56 kDa) were found by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Inhibition of the labeling of both proteins by 50% was observed in the presence of a 15‐fold molar excess of 5‐HT. Drugs and reagents affected to the same degree the binding of [3H]5‐HT to SBP (45 kDa and 56 kDa) and the binding of NAP[3H]5‐HT to the two proteins. The 45‐kDa SBP and 56‐kDa SBP covalently labeled with NAP[3H]5‐HT were analyzed by partial proteolytic digestion with either Staphylococcus aureus V8 protease or proteinase K. The generated radiolabeled peptides, separated on SDS‐PAGE from both forms of the labeled SBP, exhibited a similar pattern, suggesting their close structural similarity. Structure‐binding requirements suggest that this probe will be also useful in studying other proteins that bind 5‐HT, such as carriers of 5‐HT in the plasma membrane and vesicles, as well as some serotonergic receptors (5‐HT1p).