Serotonin binding protein: synthesis, secretion, and recycling.
Serotonin binding protein (SBP) is present in all neurectodermally derived cells that store serotonin (5-HT). Three forms of SBP have been detected (68, 56, and 45 kDa), and antibodies to SBP that interfere with the binding of 5-HT react with each of these proteins. The current experiments test two hypotheses: (a) that the 56- and 45-kDa forms of SBP are produced by posttranslational cleavage of a 68-kDa precursor molecule; and (b) that 45-kDa SBP is a constituent of serotonergic secretory vesicles. Pulse-chase experiments were carried out using medullary thyroid carcinoma cells as a model. These neurectodermally derived cells produce 5-HT and all three forms of SBP. Following pulse labeling for 20 min with L-[35S]methionine, the cells were incubated in the presence of an excess of unlabeled L-methionine for 0, 30, 60, or 90 min at 37 degrees C. Alternatively, the chase was performed under conditions (20 degrees C, inhibition of ATP generation) that delay or stop transport of newly synthesized proteins from the rough endoplasmic reticulum through the Golgi apparatus. Following incubation, the cells were washed and solubilized, and SBP was immunoprecipitated. Radioactive proteins in the immunoprecipitate were electrophoretically resolved and quantified. Immediately after the pulse, each of the three forms of SBP was found to be labeled with 35S. The relative proportions of 35S-labeled 68-, 56-, and 45-kDa SBP remained the same at each interval of chase. These proportions were not changed when the chase was carried out at 20 degrees C or under conditions that blocked the biosynthesis of ATP. These observations suggest that each form of SBP is a primary product of translation, that the smaller forms of SBP are not produced by cleavage from a larger molecule, and that the size of the primary products of translation is not altered by passage to the Golgi apparatus or a post-Golgi compartment. When secretion was induced, 45-kDa SBP, but not 56- or 68-kDa SBP, was released to the medium. When antibodies to 45-kDa SBP were added to the medium at the time secretion was induced, antibody binding sites appeared as patches on the cell surfaces. Because of these sites, cells were lysed when they were stimulated to secrete in the presence of antibodies to 45-kDa SBP and guinea pig complement.(ABSTRACT TRUNCATED AT 400 WORDS)
Tamir
H
H
Division of Neuroscience, New York State Psychiatric Institute, NY 10032.
Liu
K P
KP
Hsiung
S
S
Adlersberg
M
M
Gershon
M D
MD
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002352:Carrier Proteins / Q000276:immunology / Q000378:metabolism*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D002470:Cell Survival; D002478:Cells, Cultured; D003165:Complement System Proteins / Q000494:pharmacology; D006056:Golgi Apparatus / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D007150:Immunohistochemistry; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D012701:Serotonin / Q000378:metabolism / Q000493:pharmacokinetics; D012756:Sheep; D013462:Sulfur Radioisotopes; D013961:Thyroid Gland / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D013964:Thyroid Neoplasms / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D014407:Tumor Cells, Cultured
D000906:Antibodies; D002352:Carrier Proteins; D013462:Sulfur Radioisotopes; C025366:serotonin-binding protein; D012701:Serotonin; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D003165:Complement System Proteins; D008715:Methionine